Synthetic nanocarrier combination vaccines

ABSTRACT

Disclosed are dosage forms and related methods, that include a first population of synthetic nanocarriers that have one or more first antigens coupled to them, one or more second antigens that are not coupled to the synthetic nanocarriers, and a pharmaceutically acceptable excipient.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.provisional applications 61/348,713, filed May 26, 2010, 61/348,717,filed May 26, 2010, 61/348,728, filed May 26, 2010, and 61/358,635,filed Jun. 25, 2010, the entire contents of each of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

To either minimize the number of childhood vaccinations and/or toprovide broader immune protection against different strains of a givenpathogen, there often is a desire to combine multiple antigens in asingle dosage form, the resulting vaccine being termed a multivalentvaccine. The number of antigens that can be combined in a single dosageform may be limited by the amount of each antigen required to elicit thedesired immune response and the aqueous solubility of the antigen.

At some point, the total liquid volume of the dosage form becomes toolarge to comfortably and/or safely administer the vaccine by anintramuscular and/or subcutaneous route. This limitation is especiallynoticeable in the case of multivalent conjugate vaccines such asPrevnar™, wherein each different oligosaccharide antigen is conjugatedto a protein carrier (e.g., with 7 or 13 oligosaccharide antigensconjugated to CRM197, a non-toxic mutant of diphtheria toxin); ortetravalent Meningococcal vaccines wherein the antigens are alsoconjugated to CRM197 or other detoxified forms of diphtheria toxin.

Another limitation of existing vaccine formulations is their limitedcoverage or their physical incompatibility with one another which maypreclude simple blending of two existing vaccines to create a new,combination vaccine. For example, vaccines can consist of virus likeparticles comprising one or more antigens which self assemble or arelinked to self assembling proteins. Examples include Cervarix™ andGardasil™, which are vaccines against human papilloma virus (HPV). Bothof these vaccines target antigens derived from L1 protein of a limitednumber of HPV strains. These vaccines do not provide protection againstall strains of HPV. To expand the strain coverage of these vaccines, itis desirable to be able to admix additional viral antigens which arecompatible with the existing vaccine formulations, which provide broadercoverage and thereby create a new, expanded multivalent vaccine. Itcertain circumstances it may not be possible to simply blend inadditional conventionally produced antigens to an existing vaccinebecause of undesirable interactions between the additionalconventionally produced antigens and the existing vaccine (which maylead to precipitation, aggregation, etc.).

Therefore, what is needed are compositions and methods that couldaddress the problems noted above that are associated with producingvaccines.

SUMMARY OF THE INVENTION

In one aspect, a dosage form comprising (1) a first population ofsynthetic nanocarriers that have one or more first antigens coupled tothem, (2) one or more second antigens that are not coupled to thesynthetic nanocarriers, and (3) a pharmaceutically acceptable excipientis provided.

In one embodiment, any of the dosage forms provided further comprisesone or more adjuvants that are coupled to the synthetic nanocarriers ofthe first population of synthetic nanocarriers. In another embodiment,the one or more coupled adjuvants comprise any of the adjuvants asprovided herein. In one embodiment, the one or more adjuvants comprisePluronic® block co-polymers, specifically modified or prepared peptides,muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, RC529,bacterial toxoids, toxin fragments, agonists of Toll-Like Receptors 2,3, 4, 5, 7, 8, 9 and/or combinations thereof; adenine derivatives;immunostimulatory DNA; immunostimulatory RNA; imidazoquinoline amines,imidazopyridine amines, 6,7-fused cycloalkylimidazopyridine amines,1,2-bridged imidazoquinoline amines; imiquimod; resiquimod; type Iinterferons; poly I:C; bacterial lipopolysacccharide (LPS); VSV-G;HMGB-1; flagellin or portions or derivatives thereof; orimmunostimulatory DNA molecules comprising CpGs. In another embodiment,the one or more coupled adjuvants comprise an agonist of Toll-LikeReceptor 2, 3, 4, 7, 8 or 9. In yet another embodiment, the one or morecoupled adjuvants comprise an imidazoquinoline or oxoadenine. In stillanother embodiment, the imidazoquinoline comprises resiquimod orimiquimod.

In another embodiment, any of the dosage forms provided furthercomprises one or more adjuvants that are not coupled to the syntheticnanocarriers of the first population of synthetic nanocarriers. In oneembodiment, the one or more not coupled adjuvants comprise stimulatorsor agonists of pattern recognition receptors, mineral salts, alum, alumcombined with monphosphoryl lipid A of Enterobacteria (MPL), MPL®(AS04), AS15, saponins, QS-21, Quil-A, ISCOMs, ISCOMATRIX™, MF59™,Montanide® ISA 51, Montanide® ISA 720, AS02, liposomes and liposomalformulations, AS01, synthesized or specifically prepared microparticlesand microcarriers, bacteria-derived outer membrane vesicles of N.gonorrheae or Chlamydia trachomatis, chitosan particles, depot-formingagents, Pluronic® block co-polymers, specifically modified or preparedpeptides, muramyl dipeptide, aminoalkyl glucosaminide 4-phosphates,RC529, bacterial toxoids, toxin fragments, agonists of Toll-LikeReceptors 2, 3, 4, 5, 7, 8, 9 and/or combinations thereof; adeninederivatives; immunostimulatory DNA; immunostimulatory RNA;imidazoquinoline amines, imidazopyridine amines, 6,7-fusedcycloalkylimidazopyridine amines, 1,2-bridged imidazoquinoline amines;imiquimod; resiquimod; agonist for DC surface molecule CD40; type Iinterferons; poly I:C; bacterial lipopolysacccharide (LPS); VSV-G;HMGB-1; flagellin or portions or derivatives thereof; immunostimulatoryDNA molecules comprising CpGs; proinflammatory stimuli released fromnecrotic cells; urate crystals; activated components of the complementcascade; activated components of immune complexes; complement receptoragonists; cytokines; or cytokine receptor agonists. In anotherembodiment, the one or more not coupled adjuvants comprise alum, AS01,AS02, AS04, AS15, MPL, QS-21, a saponin, or an immunostimulatory nucleicacid comprising CpG.

In yet another embodiment of any of the dosage forms provided, the oneor more first antigens are identical to the one or more second antigens.

In a further embodiment, any of the dosage forms further comprises asecond population of synthetic nanocarriers that have one or more thirdantigens coupled to them; wherein the first and third antigens are notidentical.

In yet a further embodiment, the one or more first antigens of any ofthe dosage forms comprise a B cell antigen or a T cell antigen. In oneembodiment, the T cell antigen is a universal T cell antigen or T-helpercell antigen. In another embodiment, the one or more first antigenscomprise a B cell antigen or a T cell antigen and a a universal T cellantigen or T-helper cell antigen. In yet another embodiment, theT-helper cell antigen comprises a peptide obtained or derived fromovalbumin. In still another embodiment, the peptide obtained or derivedfrom ovalbumin comprises the sequence as set forth in SEQ ID NO: 1. Inan embodiment of any of the dosage forms, the a universal T cell antigenor T helper cell antigen is coupled by encapsulation. In anotherembodiment, the one or more second antigens of any of the dosage formscomprise a B cell antigen or a T cell antigen.

In one embodiment, any of the dosage forms provided comprises a vaccinethat comprises the second antigen that is not coupled to the syntheticnanocarriers. In another embodiment, the vaccine comprises ahapten-carrier conjugate, a virus-like particle, a synthetic nanocarriervaccine, a subunit protein vaccine, or an attenuated virus. In stillanother embodiment, the vaccine is any vaccine provided herein. In yetanother embodiment, the vaccine is against any infectious agent providedherein. In still another embodiment, the vaccine is against Anthrax;Diphtheria, Tetanus and/or Pertussis; Haemophilus influenzae type B;Hepatitis B; Hepatitis A; Hepatitis C; Herpes zoster (shingles); HumanPapillomavirus (HPV); Influenza; Japanese Encephalitis; Tick-borneEncephalitis; Measles, Mumps and/or Rubella; Meningococcal disease;Pneumococcal disease; Polio; Rabies; Rotavirus; Typhoid; Varicella;Vaccinia (Smallpox); or Yellow Fever. In a further embodiment, thevaccine comprises BIOTHRAX, DAPTACEL, INFANRIX, TRIPEDIA, TRIHIBIT,KINRIX, PEDIARIX, PENTACEL, PEDVAXHIB, ACTHIB, HIBERIX, COMVAX, HAVRIX,VAQTA, ENGERIX-B, RECOMBIVAX HB, TWINRIX, ZOSTAVAX, GARDASIL, CERVARIX,FLUARIX, FLUVIRIN, FLUZONE, FLULAVAL, AFLURIA, AGRIFLU, FLUMIST, JE-VAX,IXIARO, M-M-R II, PROQUAD, MENOMUNE, MENACTRA, MENVEO, PNEUMOVAX 23,PREVNAR, PCV13, IPOL, IMOVAX RABIES, RABAVERT, ROTATEQ, ROTARIX,DECAVAC, BOOSTRIX, ADACEL, TYPHIM VI, VIVOTIF BERNA, VARIVAX, ACAM2000or YF-VAX.

In another embodiment, the one or more first antigens and/or one or moresecond antigens are obtained or derived from any of the infectiousagents provided herein. In one embodiment, the infectious agent is avirus of the Adenoviridae, Picornaviridae, Herpesviridae,Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae orParoviridae family. In still another embodiment, the one or more firstantigens and/or one or more second antigens are obtained or derived fromadenovirus, coxsackievirus, hepatitis A virus, poliovirus, Rhinovirus,Herpes simplex virus, Varicella-zoster virus, Epstein-barr virus, Humancytomegalovirus, Human herpesvirus, Hepatitis B virus, Hepatitis Cvirus, yellow fever virus, dengue virus, West Nile virus, HIV, Influenzavirus, Measles virus, Mumps virus, Parainfluenza virus, Respiratorysyncytial virus, Human metapneumovirus, Human papillomavirus, Rabiesvirus, Rubella virus, Human bocarivus or Parvovirus B19. In yet anotherembodiment, the one or more first antigens and/or one or more secondantigens are obtained or derived from a bacteria of the Bordetella,Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila,Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella,Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio orYersinia genus. In a further embodiment, the one or more first antigensand/or one or more second antigens are obtained or derived fromBordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucellacanis, Brucella melitensis, Brucella suis, Campylobacter jejuni,Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci,Clostridium botulinum, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Francisella tularensis,Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila,Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasmapneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonasaeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonellatyphimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum,Vibrio cholerae or Yersinia pestis. In another embodiment, the one ormore first antigens and/or one or more second antigens are obtained orderived from a fungus of the Candida, Aspergillus, Cryptococcus,Histoplasma, Pneumocystis or Stachybotrys genus. In still anotherembodiment, the one or more first antigens and/or one or more secondantigens are obtained or derived from C. albicans, Aspergillusfumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcuslaurentii, Cryptococcus albidus, Cryptococcus gattii, Histoplasmacapsulatum, Pneumocystis jirovecii or Stachybotrys chartarum.

In yet another embodiment, the one or more first antigens and/or one ormore second antigens comprise or are obtained or derived from any of theantigens provided herein. In one embodiment, the antigen comprises VI,VII, E1A, E3-19K, 52K, VP1, surface antigen, 3A protein, capsid protein,nucleocapsid, surface projection, transmembrane proteins, UL6, UL18,UL35, UL38, UL19, early antigen, capsid antigen, Pp65, gB, p52, latentnuclear antigen-1, NS3, envelope protein, envelope protein E2 domain,gp120, p24, lipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef(116-145), Pol (325-355), neuraminidase, nucleocapsid protein, matrixprotein, phosphoprotein, fusion protein, hemagglutinin,hemagglutinin-neuraminidase, glycoprotein, E6, E7, envelope lipoproteinor non-structural protein (NS). In another embodiment, the one or morefirst antigens and/or one or more second antigens comprise or areobtained or derived from pertussis toxin (PT), filamentous hemagglutinin(FHA), pertactin (PRN), fimbriae (FIM 2/3), VlsE; DbpA, OspA, Hia, PrpA,MltA, L7/L12, D15, 0187, VirJ, Mdh, AfuA, L7/L12, out membrane protein,LPS, antigen type A, antigen type B, antigen type C, antigen type D,antigen type E, FliC, FliD, Cwp84, alpha-toxin, theta-toxin, fructose1,6-biphosphate-aldolase (FBA), glyceraldehydes-3-phosphatedehydrogenase (GPD), pyruvate:ferredoxin oxidoreductase (PFOR),elongation factor-G (EF-G), hypothetical protein (HP), T toxin, Toxoidantigen, capsular polysaccharide, Protein D, Mip, nucleoprotein (NP),RD1, PE35, PPE68, EsxA, EsxB, RD9, EsxV, Hsp70, lipopolysaccharide,surface antigen, Sp1, Sp2, Sp3, Glycerophosphodiester Phosphodiesterase,outer membrane protein, chaperone-usher protein, capsular protein (F1)or V protein. In yet another embodiment, the one or more first antigensand/or one or more second antigens comprise or are obtained or derivedfrom surface antigen, capsular glycoprotein, Yps3P, Hsp60, Major surfaceprotein, MsgC1, MsgC3, MsgC8, MsgC9 or SchS34.

In one embodiment of any of the dosage forms provided, the one or morefirst antigens and/or one or more second antigens comprise or areobtained or derived from one or more proteins of human papilloma virus.In another embodiment of any of the dosage forms provided, the one ormore first antigens comprise or are obtained or derived from L1 proteinof human papilloma virus, and the one or more second antigens areobtained or derived from L2 protein of human papilloma virus. In yetanother embodiment of any of the dosage forms provided, the one or morefirst antigens comprise or are obtained or derived from L2 protein ofhuman papilloma virus, and the one or more second antigens are obtainedor derived from L1 protein of human papilloma virus. In still anotherembodiment of any of the dosage forms provided, the one or more firstantigens and/or one or more second antigens comprise or are obtained orderived from one or more proteins of hepatitis B virus. In anotherembodiment of any of the dosage forms provided, the one or more firstantigens and/or one or more second antigens comprise or are obtained orderived from hepatitis B surface antigen (HBsAg). In one embodiment, theHBsAg is from strain ayw produced in Saccharomyces cerevisiae. Inanother embodiment, when the one or more first antigens are obtained orderived from hepatitis B virus, the one or more second antigens compriseor are obtained or derived from one or more proteins of human papillomavirus. In a further embodiment, when the one or more second antigens areobtained or derived from hepatitis B virus, the one or more firstantigens comprise or are obtained or derived from one or more proteinsof human papilloma virus. In one embodiment, the one or more proteins ofhuman papilloma virus is the L1 and/or L2 protein of human papillomavirus. In another embodiment of any of the dosage forms provided, theone or more first antigens and/or one or more second antigens compriseor are obtained or derived from one or more proteins of influenza virus.In one embodiment, the influenza virus is influenza A virus, H5N1 avianinfluenza virus, or H1N1 influenza A virus. In another embodiment of anyof the dosage forms provided, the one or more first antigens areobtained or derived from M2 protein of influenza A virus, and the one ormore second antigens are obtained or derived from hemagglutinin of H5N1avian influenza virus. In a further embodiment of any of the dosageforms provided, the one or more first antigens are obtained or derivedfrom hemagglutinin of H5N1 avian influenza virus, and the one or moresecond antigens are obtained or derived from M2 protein of influenza Avirus. In yet another embodiment of any of the dosage forms provided,the one or more first antigens are obtained or derived from M2 proteinof influenza A virus, and the one or more second antigens are obtainedor derived from beta-propiolactone-inactivated influenza A virus H1N1.In still another embodiment of any of the dosage forms provided, the oneor more first antigens are obtained or derived frombeta-propiolactone-inactivated influenza A virus H1N1, and the one ormore second antigens are obtained or derived from M2 protein ofinfluenza A virus.

In one embodiment of any of the dosage forms provided, thepharmaceutically acceptable excipient comprises a preservative, abuffer, saline, phosphate buffered saline, a colorant, or a stabilizer.

In another embodiment of any of the dosage forms provided, the firstsynthetic nanocarriers comprise lipid-based nanoparticles, polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles, peptide orprotein-based particles, lipid-polymer nanoparticles, spheroidalnanoparticles, cubic nanoparticles, pyramidal nanoparticles, oblongnanoparticles, cylindrical nanoparticles, or toroidal nanoparticles. Inone embodiment, the first synthetic nanocarriers comprise one or morepolymers. In another embodiment, the one or more polymers comprise apolyester. In yet another embodiment, the one or more polymers compriseor further comprise a polyester coupled to a hydrophilic polymer. Instill another embodiment, the polyester comprises a poly(lactic acid),poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone.In a further embodiment, the hydrophilic polymer comprises a polyether.In yet a further embodiment, the polyether comprises polyethyleneglycol.

In another aspect, a method comprising administering any of the dosageforms provided to a subject is provided. In one embodiment, the subjecthas or is at risk of having an infection or infectious disease. Inanother embodiment, the subject has or is at risk of having cancer.

In one embodiment of any of the methods provided, the dosage form isadministered by oral, subcutaneous, pulmonary, intranasal, intradermalor intramuscular administration. In yet another aspect, any of thedosage forms is provided for use in therapy or prophylaxis. In stillanother aspect, any of the dosage forms for use in any of the methodsprovided is provided. In yet another aspect, any of the dosage forms foruse in a method of treating or preventing cancer is provided. In afurther aspect, any of the dosage forms for use in a method of treatingor preventing infection or infectious disease is provided. In oneembodiment of any of the dosage forms, the method comprisesadministration of the dosage form by oral, subcutaneous, pulmonary,intranasal, intradermal or intramuscular administration. In stillanother aspect, use of any of the dosage forms for the manufacture of amedicament for use in any of the methods is provided.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows antibody titers in mice immunized with a combination ofNC-M2e and free hemagglutinin from H5N1 avian influenza strain(Vietnam).

FIG. 2 shows antibody titers in mice immunized with a combination ofNC-M2e and free hemagglutinin from H5N1 avian influenza strain (Vietnam)admixed with 80 μg of alum.

FIG. 3 shows antibody titers in mice immunized with a combination ofNC-M2e and beta-propiolactone-inactivated influenza A virus H1N1 (H1N1New Caledonia/20/99/IVR 116) admixed with 80 μg of alum.

FIG. 4 shows antibody titers in mice immunized with a combination ofNC-L2-peptide and HBsAg strain ayw produced in the yeast Saccharomycescerevisiae admixed with 80 μg of alum.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified materials or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting of the use of alternativeterminology to describe the present invention.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entiretyfor all purposes.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a polymer”includes a mixture of two or more such molecules, reference to “asolvent” includes a mixture of two or more such solvents, reference to“an adhesive” includes mixtures of two or more such materials, and thelike.

Introduction

The inventors have unexpectedly and surprisingly discovered that theproblems and limitations noted above can be overcome by practicing theinvention disclosed herein. In particular, the inventors haveunexpectedly discovered that it is possible to provide compositions, andrelated methods, that comprise a dosage form comprising a firstpopulation of synthetic nanocarriers that have one or more firstantigens coupled to them, one or more second antigens that are notcoupled to the synthetic nanocarriers, and a pharmaceutically acceptableexcipient.

In embodiments, the populations of synthetic nanocarriers may becombined with the one or more second antigens (which may be incorporatedin a wide variety of ways) to form dosage forms according to the presentinvention. The one or more second antigens may be provided in solutionform, suspension form, powder form, etc., and may be provided as avaccine formulation. For instance, in an embodiment, the one or moresecond antigens may be provided in the form of a hapten-carrier proteinor live attenuated virus vaccine formulation, and the population ofsynthetic nanocarriers admixed with the hapten-carrier protein or liveattenuated virus vaccine formulations form a multivalent vaccine dosageform (or increase the valency of the hapten-carrier protein or liveattenuated virus vaccine formulations). In another embodiment, thepopulation of synthetic nanocarriers may be combined with proteins takenfrom an infectious organism to form a multivalent vaccine dosage formaccording to the invention. In another embodiment, the population ofsynthetic nanocarriers may be added to another population of syntheticnanocarriers that comprise the one or more second antigens to form amultivalent synthetic nanocarrier vaccine dosage form. In otherembodiments, the population of synthetic nanocarriers may be combinedwith protein antigens in the form of virus like particles to form amultivalent vaccine dosage form according to the invention. In otherembodiments, additional antigens beyond the one or more first and/orsecond antigens can be incorporated into the dosage form (throughadmixing, and other techniques disclosed herein or knownconventionally).

In an embodiment, synthetic nanocarriers comprising one or more firstantigens and optionally a a universal T cell antigen or T helper antigenand/or an adjuvant, can be added to one or more second antigens (e.g.,an existing vaccine) to create a combination vaccine with expandedbreadth of antigen coverage.

For example, the vaccines Gardasil® and Cervarix® for protection againstHPV comprise protein antigen epitopes from the major structural proteinL1 protein derived from 4 and 2 sets of HPV strains, correspondingly.Vaccines with L1 peptide antigens from as many as 9 different HPVstrains are known. Such a vaccine with multiple peptide antigens wouldpotentially protect the individual against most, but not all, HPVstrains. If a population of synthetic nanocarriers comprising one ormore peptide epitopes from another HPV structural protein, L2, is addedto an existing L1 protein-based vaccine, broader protection from an HPVchallenge would be obtained with the potential of creating a “universalHPV vaccine.” This population of water-dispersed L2 peptide syntheticnanocarriers can simply be admixed to the existing aqueous vaccineformulation much as one would add an excipient, or diluent, to theformulation. This simple method for expanding the breadth of coverageavoids having to engineer the L2 peptide into a recombinant proteinantigen form as is conventionally done. This is illustrated in Examples1 and 2 below, which together illustrate the formation of a combinationHPV vaccine containing conventional Gardasil® augmented by syntheticnanocarriers comprising a peptide derived from L2 protein.

The inventive synthetic nanocarrier combination vaccine approach can begeneralized to include other infectious disease prophylactic ortherapeutic vaccines with less than 100% protection against the variousstrains of the infectious agent. It can also be used for prophylacticand/or therapeutic vaccines directed against non-infectious diseasetargets, such as cancer or small molecule agents. In embodiments, theinventive compositions provide for combinations of syntheticnanocarriers with existing “conventional” vaccines that can beformulated easily without the limitations of protein antigen solubilityat higher concentrations. This can reduce multivalent vaccine volumes,and enhance ease of formulation.

Examples 3-6 and 8-11 show different embodiments of the presentinvention. Examples 3 and 4 illustrate a combination vaccine ofconventional hepatitis B vaccines augmented by synthetic nanocarrierswhich comprise surface adsorbed heparin as a first antigen. Examples 5and 6 illustrate an oral combination vaccine of a conventionalanti-rotaviral vaccine augmented by synthetic nanoparticles thatcomprise peptides derived from the L2 protein of HPV. Examples 8 and 9illustrate a combination vaccine of free hemagglutinin from H5N1 avianinfluenza strain (Vietnam) augmented by synthetic nanocarriers thatcomprise M2e, OP-II T-helper peptide and R848 without or with admixedadjuvant, respectively. Example 10 illustrates a combination ofinactivated influenza A virus H1N1 vaccine and augmented with syntheticnanocarriers that comprise M2e, OP-II T-helper peptide and R848 adjuvantwith admixed alum. Example 11 illustrates a combination of recombinanthepatitis B surface antigen augmented with synthetic nanocarriers thatcomprise L2 peptide, OP-II T-helper peptide and R848 with admixed alum.The compositions exemplified in the Examples are also provided herein asare methods of their administration to a subject.

The invention will now be described in more detail below.

Definitions

“Adjuvant” means an agent that does not constitute a specific antigen,but boosts the strength and longevity of immune response to aco-administered antigen, preferably an antigen present in a dosage formtogether with the antigen, and more preferably a concomitantlyadministered antigen. Such adjuvants may include, but are not limited tostimulators of pattern recognition receptors, such as Toll-likereceptors, RIG-1 and NOD-like receptors (NLR), mineral salts, such asalum, alum combined with monphosphoryl lipid (MPL) A of Enterobacteria,such as Escherihia coli, Salmonella minnesota, Salmonella typhimurium,or Shigella flexneri or specifically with MPL® (AS04), MPL A ofabove-mentioned bacteria separately, saponins, such as QS-21, Quil-A,ISCOMs, ISCOMATRIX™, emulsions such as MF59™, Montanide® ISA 51 and ISA720, AS02 (QS21+squalene+MPL®), AS15, liposomes and liposomalformulations such as AS01, synthesized or specifically preparedmicroparticles and microcarriers such as bacteria-derived outer membranevesicles (OMV) of N. gonorrheae, Chlamydia trachomatis and others, orchitosan particles, depot-forming agents, such as Pluronic® blockco-polymers, specifically modified or prepared peptides, such as muramyldipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529, orproteins, such as bacterial toxoids or toxin fragments.

In embodiments, adjuvants comprise agonists for pattern recognitionreceptors (PRR), including, but not limited to Toll-Like Receptors(TLRs), specifically TLRs 2, 3, 4, 5, 7, 8, 9 and/or combinationsthereof. In other embodiments, adjuvants comprise agonists for Toll-LikeReceptors 3, agonists for Toll-Like Receptors 7 and 8, or agonists forToll-Like Receptor 9; preferably the recited adjuvants compriseimidazoquinolines; such as R848 (resiquimod); adenine derivatives, suchas those disclosed in U.S. Pat. No. 6,329,381 (Sumitomo PharmaceuticalCompany), US Published Patent Application 2010/0075995 to Biggadike etal., or WO 2010/018132 to Campos et al.; immunostimulatory DNA; orimmunostimulatory RNA. In specific embodiments, synthetic nanocarriersincorporate as adjuvants compounds that are agonists for toll-likereceptors (TLRs) 7 & 8 (“TLR 7/8 agonists”). Of utility are the TLR 7/8agonist compounds disclosed in U.S. Pat. No. 6,696,076 to Tomai et al.,including but not limited to imidazoquinoline amines, imidazopyridineamines, 6,7-fused cycloalkylimidazopyridine amines, and 1,2-bridgedimidazoquinoline amines. Preferred adjuvants comprise imiquimod andresiquimod. In specific embodiments, an adjuvant may be an agonist forthe DC surface molecule CD40. In certain embodiments, to stimulateimmunity rather than tolerance, a synthetic nanocarrier incorporates anadjuvant that promotes DC maturation (needed for priming of naive Tcells) and the production of cytokines, such as type I interferons,which promote antibody immune responses. In embodiments, adjuvants alsomay comprise immunostimulatory RNA molecules, such as but not limited todsRNA or poly I:poly C12U (available as Ampligen®, both poly I:C andpoly I:polyC12U being known as TLR3 stimulants), and/or those disclosedin F. Heil et al., “Species-Specific Recognition of Single-Stranded RNAvia Toll-like Receptor 7 and 8” Science 303(5663), 1526-1529 (2004); J.Vollmer et al., “Immune modulation by chemically modifiedribonucleosides and oligoribonucleotides” WO 2008033432 A2; A. Forsbachet al., “Immunostimulatory oligoribonucleotides containing specificsequence motif(s) and targeting the Toll-like receptor 8 pathway” WO2007062107 A2; E. Uhlmann et al., “Modified oligoribonucleotide analogswith enhanced immunostimulatory activity” U.S. Pat. Appl. Publ. US2006241076; G. Lipford et al., “Immunostimulatory viral RNAoligonucleotides and use for treating cancer and infections” WO2005097993 A2; G. Lipford et al., “Immunostimulatory G,U-containingoligoribonucleotides, compositions, and screening methods” WO 2003086280A2. In some embodiments, an adjuvant may be a TLR-4 agonist, such asbacterial lipopolysacccharide (LPS), VSV-G, and/or HMGB-1. In someembodiments, adjuvants may comprise TLR-5 agonists, such as flagellin,or portions or derivatives thereof, including but not limited to thosedisclosed in U.S. Pat. Nos. 6,130,082, 6,585,980, and 7,192,725. Inspecific embodiments, synthetic nanocarriers incorporate a ligand forToll-like receptor (TLR)-9, such as immunostimulatory DNA moleculescomprising CpGs, which induce type I interferon secretion, and stimulateT and B cell activation leading to increased antibody production andcytotoxic T cell responses (Krieg et al., CpG motifs in bacterial DNAtrigger direct B cell activation. Nature. 1995. 374:546-549; Chu et al.CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1(Thi) immunity. J. Exp. Med. 1997. 186:1623-1631; Lipford et al.CpG-containing synthetic oligonucleotides promote B and cytotoxic T cellresponses to protein antigen: a new class of vaccine adjuvants. Eur. J.Immunol. 1997. 27:2340-2344; Roman et al. Immunostimulatory DNAsequences function as T helper-1-promoting adjuvants. Nat. Med. 1997.3:849-854; Davis et al. CpG DNA is a potent enhancer of specificimmunity in mice immunized with recombinant hepatitis B surface antigen.J. Immunol. 1998. 160:870-876; Lipford et al., Bacterial DNA as immunecell activator. Trends Microbiol. 1998. 6:496-500; U.S. Pat. No.6,207,646 to Krieg et al.; U.S. Pat. No. 7,223,398 to Tuck et al.; U.S.Pat. No. 7,250,403 to Van Nest et al.; or U.S. Pat. No. 7,566,703 toKrieg et al.

In some embodiments, adjuvants may be proinflammatory stimuli releasedfrom necrotic cells (e.g., urate crystals). In some embodiments,adjuvants may be activated components of the complement cascade (e.g.,CD21, CD35, etc.). In some embodiments, adjuvants may be activatedcomponents of immune complexes. The adjuvants also include complementreceptor agonists, such as a molecule that binds to CD21 or CD35. Insome embodiments, the complement receptor agonist induces endogenouscomplement opsonization of the synthetic nanocarrier. In someembodiments, adjuvants are cytokines, which are small proteins orbiological factors (in the range of 5 kD-20 kD) that are released bycells and have specific effects on cell-cell interaction, communicationand behavior of other cells. In some embodiments, the cytokine receptoragonist is a small molecule, antibody, fusion protein, or aptamer.

In embodiments, at least a portion of the dose of adjuvant may becoupled to synthetic nanocarriers, preferably, all of the dose ofadjuvant is coupled to synthetic nanocarriers. In other embodiments, atleast a portion of the dose of the adjuvant is not coupled to thesynthetic nanocarriers. In embodiments, the dose of adjuvant comprisestwo or more types of adjuvants. For instance, and without limitation,adjuvants that act on different TLR receptors may be combined. As anexample, in an embodiment a TLR 7/8 agonist may be combined with a TLR 9agonist. In another embodiment, a TLR 7/8 agonist may be combined with aTLR 4 agonist. In yet another embodiment, a TLR 9 agonist may becombined with a TLR 3 agonist.

“Administering” or “administration” means providing a dosage form to asubject in a manner that is pharmacologically useful.

“Amount effective” is any amount of a composition that produces one ormore desired immune responses. This amount can be for in vitro or invivo purposes. For in vivo purposes, the amount can be one that a healthpractitioner would believe may have a clinical benefit for a subject inneed of an antibody response specific to one or more antigens. Inembodiments, therefore, an amount effective is one that a healthpractitioner would believe may generate an antibody response against theantigen(s) of the inventive compositions provided herein. Effectiveamounts can be monitored by routine methods. An amount that is effectiveto produce one or more desired immune responses can also be an amount ofa composition provided herein that produces a desired therapeuticendpoint or a desired therapeutic result. Therefore, in otherembodiments, the amount effective in one that a clinician would believewould provide a therapeutic benefit (including a prophylactic benefit)to a subject provided herein. Such subjects include those that have orare at risk of having cancer, an infection or infectious disease.

Amounts effective will depend, of course, on the particular subjectbeing treated; the severity of a condition, disease or disorder; theindividual patient parameters including age, physical condition, sizeand weight; the duration of the treatment; the nature of concurrenttherapy (if any); the specific route of administration and like factorswithin the knowledge and expertise of the health practitioner. Thesefactors are well known to those of ordinary skill in the art and can beaddressed with no more than routine experimentation. It is generallypreferred that a “maximum dose” be used, that is, the highest safe doseaccording to sound medical judgment. It will be understood by those ofordinary skill in the art, however, that a patient may insist upon alower dose or tolerable dose for medical reasons, psychological reasonsor for virtually any other reasons. The antigen(s) of any of theinventive compositions provided herein can in embodiments be in anamount effective.

“Antigen” means a B cell antigen or T cell antigen. In embodiments,antigens are coupled to the synthetic nanocarriers. In otherembodiments, antigens are not coupled to the synthetic nanocarriers. Inembodiments, dosage forms according to the invention comprise one ormore antigens, for example, one or more first antigens, one or moresecond antigens, one or more third antigens, one or more fourthantigens, and one or more additional antigens. In embodiments, antigensare coadministered with the synthetic nanocarriers. In other embodimentsantigens are not coadministered with the synthetic nanocarriers.“Type(s) of antigens” means molecules that share the same, orsubstantially the same, antigenic characteristics.

“At least a portion of the dose” means at least some part of the dose,ranging up to including all of the dose.

An “at risk” subject is one in which a health practitioner believes hasa chance of having a disease or condition provided herein including, butnot limited to, an infection, infectious disease or cancer.

“B cell antigen” means any antigen that is or recognized by and triggersan immune response in a B cell (e.g., an antigen that is specificallyrecognized by a B cell receptor on a B cell). In some embodiments, anantigen that is a T cell antigen is also a B cell antigen. In otherembodiments, the T cell antigen is not also a B cell antigen. B cellantigens include, but are not limited to, proteins, peptides, smallmolecules, and carbohydrates. In some embodiments, the B cell antigencomprises a non-protein antigen (i.e., not a protein or peptideantigen). In some embodiments, the B cell antigen comprises acarbohydrate associated with an infectious agent. In some embodiments,the B cell antigen comprises a glycoprotein or glycopeptide associatedwith an infectious agent. The infectious agent can be a bacterium,virus, fungus, protozoan, parasite or prion. In some embodiments, the Bcell antigen comprises a poorly immunogenic antigen. In someembodiments, the B cell antigen comprises an abused substance or aportion thereof. In some embodiments, the B cell antigen comprises anaddictive substance or a portion thereof. Addictive substances include,but are not limited to, nicotine, a narcotic, a cough suppressant, atranquilizer, and a sedative. In some embodiments, the B cell antigencomprises a toxin, such as a toxin from a chemical weapon or naturalsources, or a pollutant. The B cell antigen may also comprise ahazardous environmental agent. In some embodiments, the B cell antigencomprises a self antigen. In other embodiments, the B cell antigencomprises an alloantigen, an allergen, a contact sensitizer, adegenerative disease antigen, a hapten, an infectious disease antigen, acancer antigen, an atopic disease antigen, an autoimmune diseaseantigen, an addictive substance, a xenoantigen, or a metabolic diseaseenzyme or enzymatic product thereof.

“Couple” or “Coupled” or “Couples” (and the like) means to chemicallyassociate one entity (for example a moiety) with another. In someembodiments, the coupling is covalent, meaning that the coupling occursin the context of the presence of a covalent bond between the twoentities. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. In embodiments,encapsulation is a form of coupling. In embodiments, populations ofsynthetic nanocarriers have one or more antigens and/or adjuvantscoupled to them, meaning that a plurality, preferably a majority, of thesynthetic nanocarriers within the population have coupled to them one ormore antigens and/or adjuvants that are similar to one another. In otherembodiments, inventive dosage forms may comprise antigens and/oradjuvants that are not coupled to synthetic nanocarriers within apopulation of synthetic nanocarriers.

“Derived” means taken from a source and subjected to substantialmodification. For instance, a peptide or nucleic acid with a sequencewith only 50% identity to a natural peptide or nucleic acid, preferablya natural consensus peptide or nucleic acid, would be said to be derivedfrom the natural peptide or nucleic acid. Substantial modification ismodification that significantly affects the chemical or immunologicalproperties of the material in question. Derived peptides and nucleicacids can also include those with a sequence with greater than 50%identity to a natural peptide or nucleic acid sequence if said derivedpeptides and nucleic acids have altered chemical or immunologicalproperties as compared to the natural peptide or nucleic acid. Thesechemical or immunological properties comprise hydrophilicity, stability,affinity, and ability to couple with a carrier such as a syntheticnanocarrier.

“Dosage form” means a pharmacologically and/or immunologically activematerial in a medium, carrier, vehicle, or device suitable foradministration to a subject.

“Encapsulate” means to enclose within a synthetic nanocarrier,preferably enclose completely within a synthetic nanocarrier. Most orall of a substance that is encapsulated is not exposed to the localenvironment external to the synthetic nanocarrier. Encapsulation isdistinct from absorption, which places most or all of a substance on asurface of a synthetic nanocarrier, and leaves the substance exposed tothe local environment external to the synthetic nanocarrier.

“Identical” means that a substance shares one or more common chemicaland/or immunological characteristics with another substance. Forinstance, one or more antigens are identical to one or more otherantigens when both sets of antigens share one or more common chemicaland/or immunological characteristics. Substances, such as antigens, arenot identical when they fail to meet the criteria for being identical.Certain biologically active macromolecules may be described as having apercent identity with respect to one another, which is a measure of thematching of their sequences, as is conventionally known in the art. Suchbiologically active macromolecules are identical within the scope ofthis invention when they share greater than 20% identity, preferablygreater than 30% identity, preferably greater than 40% identity,preferably greater than 50% identity, preferably greater than 60%identity, preferably greater than 70% identity, preferably greater than80% identity, or preferably greater than 90% identity, with one another.

An “infection” or “infectious disease” is any condition or diseasecaused by a microorganism, pathogen or other agent, such as a bacterium,fungus, prion or virus.

“Isolated nucleic acid” means a nucleic acid that is separated from itsnative environment and present in sufficient quantity to permit itsidentification or use. An isolated nucleic acid may be one that is (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. Any of the nucleic acids provided herein may be isolated. Insome embodiments, the antigens in the compositions provided herein arepresent in the form of an isolated nucleic acid, such as an isolatednucleic acid that encodes an antigenic peptide, polypeptide or protein.

“Isolated peptide, polypeptide or protein” means the polypeptide (orpeptide or protein) is separated from its native environment and presentin sufficient quantity to permit its identification or use. This means,for example, the polypeptide (or peptide or protein) may be (i)selectively produced by expression cloning or (ii) purified as bychromatography or electrophoresis. Isolated peptides, proteins orpolypeptides may be, but need not be, substantially pure. Because anisolated peptide, polypeptide or protein may be admixed with apharmaceutically acceptable carrier in a pharmaceutical preparation, thepolypeptide (or peptide or protein) may comprise only a small percentageby weight of the preparation. The polypeptide (or peptide or protein) isnonetheless isolated in that it has been separated from the substanceswith which it may be associated in living systems, i.e., isolated fromother proteins (or peptides or polypeptides). Any of the peptides,polypeptides or proteins provided herein may be isolated. In someembodiments, the antigens in the compositions provided herein arepeptides, polypeptides or proteins.

“Maximum dimension of a synthetic nanocarrier” means the largestdimension of a nanocarrier measured along any axis of the syntheticnanocarrier. “Minimum dimension of a synthetic nanocarrier” means thesmallest dimension of a synthetic nanocarrier measured along any axis ofthe synthetic nanocarrier. For example, for a spheroidal syntheticnanocarrier, the maximum and minimum dimension of a syntheticnanocarrier would be substantially identical, and would be the size ofits diameter. Similarly, for a cuboidal synthetic nanocarrier, theminimum dimension of a synthetic nanocarrier would be the smallest ofits height, width or length, while the maximum dimension of a syntheticnanocarrier would be the largest of its height, width or length. In anembodiment, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 100 nm. In an embodiment, a maximum dimension ofat least 75%, preferably at least 80%, more preferably at least 90%, ofthe synthetic nanocarriers in a sample, based on the total number ofsynthetic nanocarriers in the sample, is equal to or less than 5 μm.Preferably, a minimum dimension of at least 75%, preferably at least80%, more preferably at least 90%, of the synthetic nanocarriers in asample, based on the total number of synthetic nanocarriers in thesample, is greater than 110 nm, more preferably greater than 120 nm,more preferably greater than 130 nm, and more preferably still greaterthan 150 nm. Aspects ratios of the maximum and minimum dimensions ofinventive synthetic nanocarriers may vary depending on the embodiment.For instance, aspect ratios of the maximum to minimum dimensions of thesynthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from1:1 to 100,000:1, more preferably from 1:1 to 1000:1, still preferablyfrom 1:1 to 100:1, and yet more preferably from 1:1 to 10:1. Preferably,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample isequal to or less than 3 μm, more preferably equal to or less than 2 μm,more preferably equal to or less than 1 μm, more preferably equal to orless than 800 nm, more preferably equal to or less than 600 nm, and morepreferably still equal to or less than 500 nm. In preferred embodiments,a maximum dimension of at least 75%, preferably at least 80%, morepreferably at least 90%, of the synthetic nanocarriers in a sample,based on the total number of synthetic nanocarriers in the sample, isequal to or greater than 100 nm, more preferably equal to or greaterthan 120 nm, more preferably equal to or greater than 130 nm, morepreferably equal to or greater than 140 nm, and more preferably stillequal to or greater than 150 nm. Measurement of synthetic nanocarriersizes is obtained by suspending the synthetic nanocarriers in a liquid(usually aqueous) media and using dynamic light scattering (e.g. using aBrookhaven ZetaPALS instrument).

“Obtained” means taken from a source without substantial modification.Substantial modification is modification that significantly affects thechemical or immunological properties of the material in question. Forexample, as a non-limiting example, a peptide or nucleic acid with asequence with greater than 90%, preferably greater than 95%, preferablygreater than 97%, preferably greater than 98%, preferably greater than99%, preferably 100%, identity to a natural peptide or nucleotidesequence, preferably a natural consensus peptide or nucleotide sequence,and chemical and/or immunological properties that are not significantlydifferent from the natural peptide or nucleic acid, would be said to beobtained from the natural peptide or nucleotide sequence. These chemicalor immunological properties comprise hydrophilicity, stability,affinity, and ability to couple with a carrier such as a syntheticnanocarrier.

“Pharmaceutically acceptable carrier(s) or excipient(s)” means materialsthat are contained within the dosage form, but do not contributesubstantially to the primary pharmacological activity of the dosageform. In embodiments, the materials are pharmacologically inactive. Inembodiments, pharmaceutically acceptable excipients comprisepreservatives, buffers, saline, or phosphate buffered saline, colorants,or stabilizers. Pharmaceutically acceptable excipients comprise avariety of materials known in the art, including but not limited tosaccharides (such as glucose, lactose, and the like), preservatives suchas antimicrobial agents, reconstitution aids, colorants, saline (such asphosphate buffered saline), and buffers.

“Population” means a defined group of synthetic nanocarriers that shareone or more common physical or chemical characteristics. Common physicalor chemical characteristics may comprise having a common coupledantigen(s), common coupled adjuvant(s), common materials making up thebulk nanocarrier, a common shape, a common particle size, and the like.Multiple populations of synthetic nanocarriers may be identified, forexample a first population, a second population, a third population, afourth population, and the like.

“Subject” means animals, including warm blooded mammals such as humansand primates; avians; domestic household or farm animals such as cats,dogs, sheep, goats, cattle, horses and pigs; laboratory animals such asmice, rats and guinea pigs; fish; reptiles; zoo and wild animals; andthe like.

“Synthetic nanocarrier(s)” means a discrete object that is not found innature, and that possesses at least one dimension that is less than orequal to 5 microns in size. Albumin nanoparticles are generally includedas synthetic nanocarriers, however in certain embodiments the syntheticnanocarriers do not comprise albumin nanoparticles. In embodiments,inventive synthetic nanocarriers do not comprise chitosan.

A synthetic nanocarrier can be, but is not limited to, one or aplurality of lipid-based nanoparticles (e.g. liposomes) (also referredto herein as lipid nanoparticles, i.e., nanoparticles where the majorityof the material that makes up their structure are lipids), polymericnanoparticles, metallic nanoparticles, surfactant-based emulsions,dendrimers, buckyballs, nanowires, virus-like particles (i.e., particlesthat are primarily made up of viral structural proteins but that are notinfectious or have low infectivity), peptide or protein-based particles(also referred to herein as protein particles, i.e., particles where themajority of the material that makes up their structure are peptides orproteins) (such as albumin nanoparticles) and/or nanoparticles that aredeveloped using a combination of nanomaterials such as lipid-polymernanoparticles. Synthetic nanocarriers may be a variety of differentshapes, including but not limited to spheroidal, cuboidal, pyramidal,oblong, cylindrical, toroidal, and the like. Synthetic nanocarriersaccording to the invention comprise one or more surfaces, including butnot limited to internal surfaces (surfaces generally facing an interiorportion of the synthetic nanocarrier) and external surfaces (surfacesgenerally facing an external environment of the synthetic nanocarrier).Exemplary synthetic nanocarriers that can be adapted for use in thepractice of the present invention comprise: (1) the biodegradablenanoparticles disclosed in U.S. Pat. No. 5,543,158 to Gref et al., (2)the polymeric nanoparticles of Published US Patent Application20060002852 to Saltzman et al., (3) the lithographically constructednanoparticles of Published US Patent Application 20090028910 to DeSimoneet al., (4) the disclosure of WO 2009/051837 to von Andrian et al., (5)the nanoparticles disclosed in Published US Patent Application2008/0145441 to Penades et al., (6) the protein nanoparticles disclosedin Published US Patent Application 20090226525 to de los Rios et al.,(7) the virus-like particles disclosed in published US PatentApplication 20060222652 to Sebbel et al., (8) the nucleic acid coupledvirus-like particles disclosed in published US Patent Application20060251677 to Bachmann et al., (9) the virus-like particles disclosedin WO2010047839A1 or WO2009106999A2, or (10) the nanoprecipitatednanoparticles disclosed in P. Paolicelli et al., “Surface-modifiedPLGA-based Nanoparticles that can Efficiently Associate and DeliverVirus-like Particles” Nanomedicine. 5(6):843-853 (2010). In embodiments,synthetic nanocarriers may possess an aspect ratio greater than 1:1,1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

Synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface with hydroxyl groups thatactivate complement or alternatively comprise a surface that consistsessentially of moieties that are not hydroxyl groups that activatecomplement. In a preferred embodiment, synthetic nanocarriers accordingto the invention that have a minimum dimension of equal to or less thanabout 100 nm, preferably equal to or less than 100 nm, do not comprise asurface that substantially activates complement or alternativelycomprise a surface that consists essentially of moieties that do notsubstantially activate complement. In a more preferred embodiment,synthetic nanocarriers according to the invention that have a minimumdimension of equal to or less than about 100 nm, preferably equal to orless than 100 nm, do not comprise a surface that activates complement oralternatively comprise a surface that consists essentially of moietiesthat do not activate complement. In embodiments, synthetic nanocarriersexclude virus-like particles. In embodiments, when syntheticnanocarriers comprise virus-like particles, the virus-like particlescomprise non-natural adjuvant (meaning that the VLPs comprise anadjuvant other than naturally occurring RNA generated during theproduction of the VLPs). In embodiments, synthetic nanocarriers maypossess an aspect ratio greater than 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5,1:7, or greater than 1:10.

“T cell antigen” means any antigen that is recognized by and triggers animmune response in a T cell (e.g., an antigen that is specificallyrecognized by a T cell receptor on a T cell or an NKT cell viapresentation of the antigen or portion thereof bound to a Class I orClass II major histocompatability complex molecule (MHC), or bound to aCD1 complex. In some embodiments, an antigen that is a T cell antigen isalso a B cell antigen. In other embodiments, the T cell antigen is notalso a B cell antigen. T cell antigens generally are proteins orpeptides. T cell antigens may be an antigen that stimulates a CD8+ Tcell response, a CD4+ T cell response, or both. The nanocarriers,therefore, in some embodiments can effectively stimulate both types ofresponses.

In some embodiments the T cell antigen is a ‘universal’ T cell antigen,or T cell memory antigen, (i.e., one to which a subject has apre-existing memory and that can be used to boost T cell help to anunrelated antigen, for example an unrelated B cell antigen). Universal Tcell antigens include tetanus toxoid, as well as one or more peptidesderived from tetanus toxoid, Epstein-Barr virus, or influenza virus.Universal T cell antigens also include a components of influenza virus,such as hemagglutinin, neuraminidase, or nuclear protein, or one or morepeptides derived therefrom. In some embodiments, the universal T cellantigen is not one that is presented in a complex with a MHC molecule.In some embodiments, the universal T cell antigen is not complexed witha MHC molecule for presentation to a T helper cell. Accordingly, in someembodiments, the universal T cell antigen is not a T helper cellantigen. However, in other embodiments, the universal T cell antigen isa T helper cell antigen.

In embodiments, a T-helper cell antigen may comprise one or morepeptides obtained or derived from tetanus toxoid, Epstein-Barr virus,influenza virus, respiratory syncytial virus, measles virus, mumpsvirus, rubella virus, cytomegalovirus, adenovirus, diphtheria toxoid, ora PADRE peptide (known from the work of Sette et al. U.S. Pat. No.7,202,351). In other embodiments, a T-helper cell antigen may compriseovalbumin or a peptide obtained or derived therefrom. Preferably, theovalbumin comprises the amino acid sequence as set forth in AccessionNo. AAB59956, NP_(—)990483.1, AAA48998, or CAA2371. In otherembodiments, the peptide obtained or derived from ovalbumin comprisesthe following amino acid sequence:H-Ile-Ser-Gln-Ala-Val-His-Ala-Ala-His-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg-OH(SEQ ID NO: 1). In other embodiments, a T-helper cell antigen maycomprise one or more lipids, or glycolipids, including but not limitedto: α-galactosylceramide (α-GalCer), α-linked glycosphingolipids (fromSphingomonas spp.), galactosyl diacylglycerols (from Borreliaburgdorferi), lypophosphoglycan (from Leishmania donovani), andphosphatidylinositol tetramannoside (PIM4) (from Mycobacterium leprae).For additional lipids and/or glycolipids useful as T-helper cellantigen, see V. Cerundolo et al., “Harnessing invariant NKT cells invaccination strategies.” Nature Rev Immun, 9:28-38 (2009).

In embodiments, CD4+ T-cell antigens may be derivatives of a CD4+ T-cellantigen that is obtained from a source, such as a natural source. Insuch embodiments, CD4+ T-cell antigen sequences, such as those peptidesthat bind to MHC II, may have at least 70%, 80%, 90%, or 95% identity tothe antigen obtained from the source. In embodiments, the T cellantigen, preferably a universal T cell antigen or T-helper cell antigen,may be coupled to, or uncoupled from, a synthetic nanocarrier. In someembodiments, the universal T cell antigen or T-helper cell antigen isencapsulated in the synthetic nanocarriers of the inventivecompositions.

“Vaccine” means a composition of matter that improves the immuneresponse to a particular pathogen or disease. A vaccine typicallycontains factors that stimulate a subject's immune system to recognize aspecific antigen as foreign and eliminate it from the subject's body. Avaccine also establishes an immunologic ‘memory’ so the antigen will bequickly recognized and responded to if a person is re-challenged.Vaccines can be prophylactic (for example to prevent future infection byany pathogen), or therapeutic (for example a vaccine against a tumorspecific antigen for the treatment of cancer). In embodiments, a vaccinemay comprise dosage forms according to the invention. In otherembodiments, the inventive dosage form may comprise a vaccine comprisingthe second antigen that is not coupled to the synthetic nanocarriers.Vaccines according to the invention may comprise a hapten-carrierconjugate, a virus-like particle, a synthetic nanocarrier vaccine, asubunit protein vaccine, or an attenuated virus. In some embodiments,the vaccine comprises any of the vaccines, including the commerciallyavailable vaccines, described herein.

Inventive Compositions

A wide variety of synthetic nanocarriers can be used according to theinvention. In some embodiments, synthetic nanocarriers are spheres orspheroids. In some embodiments, synthetic nanocarriers are flat orplate-shaped. In some embodiments, synthetic nanocarriers are cubes orcuboidal. In some embodiments, synthetic nanocarriers are ovals orellipses. In some embodiments, synthetic nanocarriers are cylinders,cones, or pyramids.

In some embodiments, it is desirable to use a population of syntheticnanocarriers that is relatively uniform in terms of size, shape, and/orcomposition so that each synthetic nanocarrier has similar properties.For example, at least 80%, at least 90%, or at least 95% of thesynthetic nanocarriers, based on the total number of syntheticnanocarriers, may have a minimum dimension or maximum dimension thatfalls within 5%, 10%, or 20% of the average diameter or averagedimension of the synthetic nanocarriers. In some embodiments, apopulation of synthetic nanocarriers may be heterogeneous with respectto size, shape, and/or composition.

Synthetic nanocarriers can be solid or hollow and can comprise one ormore layers. In some embodiments, each layer has a unique compositionand unique properties relative to the other layer(s). To give but oneexample, synthetic nanocarriers may have a core/shell structure, whereinthe core is one layer (e.g. a polymeric core) and the shell is a secondlayer (e.g. a lipid bilayer or monolayer). Synthetic nanocarriers maycomprise a plurality of different layers.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more lipids. In some embodiments, a synthetic nanocarrier maycomprise a liposome. In some embodiments, a synthetic nanocarrier maycomprise a lipid bilayer. In some embodiments, a synthetic nanocarriermay comprise a lipid monolayer. In some embodiments, a syntheticnanocarrier may comprise a micelle. In some embodiments, a syntheticnanocarrier may comprise a core comprising a polymeric matrix surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.). In someembodiments, a synthetic nanocarrier may comprise a non-polymeric core(e.g., metal particle, quantum dot, ceramic particle, bone particle,viral particle, proteins, nucleic acids, carbohydrates, etc.) surroundedby a lipid layer (e.g., lipid bilayer, lipid monolayer, etc.).

In some embodiments, synthetic nanocarriers can comprise one or morepolymers. In some embodiments, such a polymer can be surrounded by acoating layer (e.g., liposome, lipid monolayer, micelle, etc.). In someembodiments, various elements of the synthetic nanocarriers can becoupled with the polymer.

In some embodiments, an immunofeature surface, targeting moiety,antigen, adjuvant and/or oligonucleotide can be covalently associatedwith a polymeric matrix. In some embodiments, covalent association ismediated by a linker. In some embodiments, an immunofeature surface,targeting moiety, antigen, adjuvant and/or oligonucleotide can benoncovalently associated with a polymeric matrix. For example, in someembodiments, an immunofeature surface, targeting moiety, antigen,adjuvant and/or oligonucleotide can be adsorbed upon, encapsulatedwithin, surrounded by, and/or dispersed throughout a polymeric matrix.Alternatively or additionally, an immunofeature surface, targetingmoiety, antigen, adjuvant and/or nucleotide can be associated with apolymeric matrix by hydrophobic interactions, charge interactions, vander Waals forces, etc.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventionally. In general, a polymeric matrixcomprises one or more polymers. Polymers may be natural or unnatural(synthetic) polymers. Polymers may be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers may berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the present invention include,but are not limited to polyethylenes, polycarbonates (e.g.poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacic anhydride)),polypropylfumerates, polyamides (e.g. polycaprolactam), polyacetals,polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(β-hydroxyalkanoate)), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers which have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. §177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid); polycaprolactone (e.g.,poly(1,3-dioxan-2one)); polyvalerolactone; polyanhydrides (e.g.,poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol);polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, a synthetic nanocarrier comprising a hydrophilic polymericmatrix generates a hydrophilic environment within the syntheticnanocarrier. In some embodiments, polymers can be hydrophobic. In someembodiments, a synthetic nanocarrier comprising a hydrophobic polymericmatrix generates a hydrophobic environment within the syntheticnanocarrier. Selection of the hydrophilicity or hydrophobicity of thepolymer may have an impact on the nature of materials that areincorporated (e.g. coupled) within the synthetic nanocarrier.

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers may be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments may be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al., or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group may be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g. DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines. In embodiments, the inventive synthetic nanocarriers maynot comprise (or may exclude) cationic polymers.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al.,1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc.,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al.,2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am. Chem. Soc.,123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J.Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanocarriers may comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, the synthetic nanocarriers comprise one or morepolymers. The polymeric synthetic nanocarriers, therefore, can alsoinclude those described in WO publication WO2009/051837 by Von Andrianet al., including, but not limited to those, with one or morehydrophilic components. Preferably, the one or more polymers comprise apolyester, such as a poly(lactic acid), poly(glycolic acid),poly(lactic-co-glycolic acid), or polycaprolactone. More preferably, theone or more polymers comprise or further comprise a polyester coupled toa hydrophilic polymer, such as a polyether. In embodiments, thepolyether comprises polyethylene glycol. Still more preferably, the oneor more polymers comprise a polyester and a polyester coupled to ahydrophilic polymer, such as a polyether. In other embodiments, the oneor more polymers are coupled to one or more antigens and/or one or moreadjuvants. In embodiments, at least some of the polymers are coupled tothe antigen(s) and/or at least some of the polymers are coupled to theadjuvant(s). Preferably, when there are more than one type of polymer,one of the types of polymer is coupled to the antigen(s). Inembodiments, one of the other types of polymer is coupled to theadjuvant(s). For example, in embodiments, when the nanocarriers comprisea polyester and a polyester coupled to a hydrophilic polymer, such as apolyether, the polyester is coupled to the adjuvant, while the polyestercoupled to the hydrophilic polymer, such as a polyether, is coupled tothe antigen(s). In embodiments, where the nanocarriers comprise a Thelper cell antigen, the T helper cell antigen can be encapsulated inthe nanocarrier.

In some embodiments, synthetic nanocarriers do not comprise a polymericcomponent. In some embodiments, synthetic nanocarriers may comprisemetal particles, quantum dots, ceramic particles, etc. In someembodiments, a non-polymeric synthetic nanocarrier is an aggregate ofnon-polymeric components, such as an aggregate of metal atoms (e.g.,gold atoms).

In some embodiments, synthetic nanocarriers may optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanocarriers with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanocarriers in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxomer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component may be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity may be used in the production ofsynthetic nanocarriers to be used in accordance with the presentinvention.

In some embodiments, synthetic nanocarriers may optionally comprise oneor more carbohydrates. Carbohydrates may be natural or synthetic. Acarbohydrate may be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellbiose, mannose, xylose,arabinose, glucoronic acid, galactoronic acid, mannuronic acid,glucosamine, galatosamine, and neuramic acid. In certain embodiments, acarbohydrate is a polysaccharide, including but not limited to pullulan,cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose(HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran,cyclodextran, glycogen, starch, hydroxyethylstarch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, inulin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In embodiments, the inventivesynthetic nanocarriers do not comprise (or specifically exclude)carbohydrates, such as a polysaccharide. In certain embodiments, thecarbohydrate may comprise a carbohydrate derivative such as a sugaralcohol, including but not limited to mannitol, sorbitol, xylitol,erythritol, maltitol, and lactitol.

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients,such as preservatives, buffers, saline, or phosphate buffered saline.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. Typical inventive compositions may comprise inorganic or organicbuffers (e.g., sodium or potassium salts of phosphate, carbonate,acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid,sodium or potassium hydroxide, salts of citrate or acetate, amino acidsand their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol),surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10nonyl phenol, sodium desoxycholate), solution and/or cryo/lyostabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmoticadjustment agents (e.g., salts or sugars), antibacterial agents (e.g.,benzoic acid, phenol, gentamicin), antifoaming agents (e.g.,polydimethylsilozone), preservatives (e.g., thimerosal,2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustmentagents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol). In an embodiment, inventive synthetic nanocarriers aresuspended in sterile saline solution for injection together with apreservative.

In embodiments, when preparing synthetic nanocarriers as carriers forantigens or adjuvants for use in vaccines, methods for coupling theantigens or adjuvants to the synthetic nanocarriers may be useful. Ifthe antigens or adjuvant is a small molecule it may be of advantage toattach the antigens or adjuvant to a polymer prior to the assembly ofthe synthetic nanocarriers. In embodiments, it may also be an advantageto prepare the synthetic nanocarriers with surface groups that are usedto couple the antigens or adjuvant to the synthetic nanocarrier throughthe use of these surface groups rather than attaching the antigens oradjuvant to a polymer and then using this polymer conjugate in theconstruction of synthetic nanocarriers.

In certain embodiments, the coupling can be a covalent linker. Inembodiments, peptides according to the invention can be covalentlycoupled to the external surface via a 1,2,3-triazole linker formed bythe 1,3-dipolar cycloaddition reaction of azido groups on the surface ofthe nanocarrier with antigen or adjuvant containing an alkyne group orby the 1,3-dipolar cycloaddition reaction of alkynes on the surface ofthe nanocarrier with antigens or adjuvants containing an azido group.Such cycloaddition reactions are preferably performed in the presence ofa Cu(I) catalyst along with a suitable Cu(I)-ligand and a reducing agentto reduce Cu(II) compound to catalytic active Cu(I) compound. ThisCu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) can also be referredas the click reaction.

Additionally, the covalent coupling may comprise a covalent linker thatcomprises an amide linker, a disulfide linker, a thioether linker, ahydrazone linker, a hydrazide linker, an imine or oxime linker, an ureaor thiourea linker, an amidine linker, an amine linker, and asulfonamide linker.

An amide linker is formed via an amide bond between an amine on onecomponent such as the antigen or adjuvant with the carboxylic acid groupof a second component such as the nanocarrier. The amide bond in thelinker can be made using any of the conventional amide bond formingreactions with suitably protected amino acids or antigens or adjuvantsand activated carboxylic acid such N-hydroxysuccinimide-activated ester.

A disulfide linker is made via the formation of a disulfide (S—S) bondbetween two sulfur atoms of the form, for instance, of R1-S—S—R2. Adisulfide bond can be formed by thiol exchange of an antigen or adjuvantcontaining thiol/mercaptan group (—SH) with another activated thiolgroup on a polymer or nanocarrier or a nanocarrier containingthiol/mercaptan groups with a antigen or adjuvant containing activatedthiol group.

A triazole linker, specifically a 1,2,3-triazole of the form

wherein R1 and R2 may be any chemical entities, is made by the1,3-dipolar cycloaddition reaction of an azide attached to a firstcomponent such as the nanocarrier with a terminal alkyne attached to asecond component such as the peptide. The 1,3-dipolar cycloadditionreaction is performed with or without a catalyst, preferably withCu(I)-catalyst, which links the two components through a 1,2,3-triazolefunction. This chemistry is described in detail by Sharpless et al.,Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al, Chem.Rev., 2008, 108(8), 2952-3015 and is often referred to as a “click”reaction or CuAAC.

In embodiments, a polymer containing an azide or alkyne group, terminalto the polymer chain is prepared. This polymer is then used to prepare asynthetic nanocarrier in such a manner that a plurality of the alkyne orazide groups are positioned on the surface of that nanocarrier.Alternatively, the synthetic nanocarrier can be prepared by anotherroute, and subsequently functionalized with alkyne or azide groups. Theantigen or adjuvantis prepared with the presence of either an alkyne (ifthe polymer contains an azide) or an azide (if the polymer contains analkyne) group. The antigen or adjuvant is then allowed to react with thenanocarrier via the 1,3-dipolar cycloaddition reaction with or without acatalyst which covalently couples the antigen or adjuvant to theparticle through the 1,4-disubstituted 1,2,3-triazole linker.

A thioether linker is made by the formation of a sulfur-carbon(thioether) bond in the form, for instance, of R1-S—R2. Thioether can bemade by either alkylation of a thiol/mercaptan (—SH) group on onecomponent such as the antigen or adjuvant with an alkylating group suchas halide or epoxide on a second component such as the nanocarrier.Thioether linkers can also be formed by Michael addition of athiol/mercaptan group on one component such as a antigen or adjuvant toan electron-deficient alkene group on a second component such as apolymer containing a maleimide group or vinyl sulfone group as theMichael acceptor. In another way, thioether linkers can be prepared bythe radical thiol-ene reaction of a thiol/mercaptan group on onecomponent such as a antigen or adjuvant with an alkene group on a secondcomponent such as a polymer or nanocarrier.

A hydrazone linker is made by the reaction of a hydrazide group on onecomponent such as the antigen or adjuvant with an aldehyde/ketonechemistrygroup on the second component such as the nanocarrier.

A hydrazide linker is formed by the reaction of a hydrazine group on onecomponent such as the antigen or adjuvant with a carboxylic acid groupon the second component such as the nanocarrier. Such reaction isgenerally performed using chemistry similar to the formation of amidebond where the carboxylic acid is activated with an activating reagent.

An imine or oxime linker is formed by the reaction of an amine orN-alkoxyamine (or aminooxy) group on one component such as the antigenor adjuvant with an aldehyde or ketone group on the second componentsuch as the nanocarrier.

An urea or thiourea linker is prepared by the reaction of an amine groupon one component such as the antigen or adjuvant with an isocyanate orthioisocyanate group on the second component such as the nanocarrier.

An amidine linker is prepared by the reaction of an amine group on onecomponent such as the antigen or adjuvant with an imidoester group onthe second component such as the nanocarrier.

An amine linker is made by the alkylation reaction of an amine group onone component such as the antigen or adjuvant with an alkylating groupsuch as halide, epoxide, or sulfonate ester group on the secondcomponent such as the nanocarrier. Alternatively, an amine linker canalso be made by reductive amination of an amine group on one componentsuch as the antigen or adjuvant with an aldehyde or ketone group on thesecond component such as the nanocarrier with a suitable reducingreagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

A sulfonamide linker is made by the reaction of an amine group on onecomponent such as the antigen or adjuvant with a sulfonyl halide (suchas sulfonyl chloride) group on the second component such as thenanocarrier.

A sulfone linker is made by Michael addition of a nucleophile to a vinylsulfone. Either the vinyl sulfone or the nucleophile may be on thesurface of the nanoparticle or attached to the antigen or adjuvant.

The antigen or adjuvant can also be conjugated to the nanocarrier vianon-covalent conjugation methods. For examples, a negative chargedantigen or adjuvant can be conjugated to a positive charged nanocarrierthrough electrostatic adsorption. An antigen or adjuvant containing ametal ligand can also be conjugated to a nanocarrier containing a metalcomplex via a metal-ligand complex.

In embodiments, an antigen or adjuvant can be attached to a polymer, forexample polylactic acid-block-polyethylene glycol, prior to the assemblyof the synthetic nanocarrier or the synthetic nanocarrier can be formedwith reactive or activatible groups on its surface. In the latter case,the antigen or adjuvant may be prepared with a group which is compatiblewith the attachment chemistry that is presented by the syntheticnanocarrier's surface. In other embodiments, a peptide antigen can beattached to VLPs or liposomes using a suitable linker. A linker is acompound or reagent that capable of coupling two molecules together. Inan embodiment, the linker can be a homobifuntional or heterobifunctionalreagent as described in Hermanson, 2008. For example, an VLP or liposomesynthetic nanocarrier containing a carboxylic group on the surface canbe treated with a homobifunctional linker, adipic dihydrazide (ADH), inthe presence of EDC to form the corresponding synthetic nanocarrier withthe ADH linker. The resulting ADH linked synthetic nanocarrier is thenconjugated with a peptide antigen containing an acid group via the otherend of the ADH linker on NC to produce the corresponding VLP or liposomepeptide conjugate.

For detailed descriptions of available conjugation methods, seeHermanson G T “Bioconjugate Techniques”, 2nd Edition Published byAcademic Press, Inc., 2008. In addition to covalent attachment theantigen or adjuvant can be coupled by adsorption to a pre-formedsynthetic nanocarrier or it can be coupled by encapsulation during theformation of the synthetic nanocarrier.

Methods of Making and Using the Inventive Dosage Forms and RelatedMethods

Synthetic nanocarriers may be prepared using a wide variety of methodsknown in the art. For example, synthetic nanocarriers can be formed bymethods as nanoprecipitation, flow focusing fluidic channels, spraydrying, single and double emulsion solvent evaporation, solventextraction, phase separation, milling, microemulsion procedures,microfabrication, nanofabrication, sacrificial layers, simple andcomplex coacervation, and other methods well known to those of ordinaryskill in the art. Alternatively or additionally, aqueous and organicsolvent syntheses for monodisperse semiconductor, conductive, magnetic,organic, and other nanomaterials have been described (Pellegrino et al.,2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; andTrindade et al., 2001, Chem. Mat., 13:3843). Additional methods havebeen described in the literature (see, e.g., Doubrow, Ed.,“Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRC Press,Boca Raton, 1992; Mathiowitz et al., 1987, J. Control. Release, 5:13;Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz etal., 1988, J. Appl. Polymer Sci., 35:755, U.S. Pat. Nos. 5,578,325 and6,007,845; P. Paolicelli et al., “Surface-modified PLGA-basedNanoparticles that can Efficiently Associate and Deliver Virus-likeParticles” Nanomedicine. 5(6):843-853 (2010)).

Various materials may be encapsulated into synthetic nanocarriers asdesirable using a variety of methods including but not limited to C.Astete et al., “Synthesis and characterization of PLGA nanoparticles” J.Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K.Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide)Nanoparticles: Preparation, Properties and Possible Applications in DrugDelivery” Current Drug Delivery 1:321-333 (2004); C. Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymericnanoparticles” Nanomedicine 2:8-21 (2006); P. Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can EfficientlyAssociate and Deliver Virus-like Particles” Nanomedicine. 5(6):843-853(2010). Other methods suitable for encapsulating materials, such asoligonucleotides, into synthetic nanocarriers may be used, includingwithout limitation methods disclosed in U.S. Pat. No. 6,632,671 to Unger(Oct. 14, 2003).

In certain embodiments, synthetic nanocarriers are prepared by ananoprecipitation process or spray drying. Conditions used in preparingsynthetic nanocarriers may be altered to yield particles of a desiredsize or property (e.g., hydrophobicity, hydrophilicity, externalmorphology, “stickiness,” shape, etc.). The method of preparing thesynthetic nanocarriers and the conditions (e.g., solvent, temperature,concentration, air flow rate, etc.) used may depend on the materials tobe coupled to the synthetic nanocarriers and/or the composition of thepolymer matrix.

If particles prepared by any of the above methods have a size rangeoutside of the desired range, particles can be sized, for example, usinga sieve.

Elements of the inventive synthetic nanocarriers (such as moieties ofwhich an immunofeature surface is comprised, targeting moieties,polymeric matrices, antigens, adjuvants, and the like) may be coupled tothe overall synthetic nanocarrier, e.g., by one or more covalent bonds,or may be coupled by means of one or more linkers. Additional methods offunctionalizing synthetic nanocarriers may be adapted from Published USPatent Application 2006/0002852 to Saltzman et al., Published US PatentApplication 2009/0028910 to DeSimone et al., or Published InternationalPatent Application WO/2008/127532 A1 to Murthy et al.

Alternatively or additionally, synthetic nanocarriers can be coupled toimmunofeature surfaces, targeting moieties, adjuvants, various antigens,and/or other elements directly or indirectly via non-covalentinteractions. In non-covalent embodiments, the non-covalent coupling ismediated by non-covalent interactions including but not limited tocharge interactions, affinity interactions, metal coordination, physicaladsorption, host-guest interactions, hydrophobic interactions, TTstacking interactions, hydrogen bonding interactions, van der Waalsinteractions, magnetic interactions, electrostatic interactions,dipole-dipole interactions, and/or combinations thereof. Such couplingsmay be arranged to be on an external surface or an internal surface ofan inventive synthetic nanocarrier. In embodiments, encapsulation and/orabsorption is a form of coupling.

A wide variety of the one or more second antigens (or additionalantigens that are not coupled to the population of syntheticnanocarriers) may be incorporated into the dosage form, and may beincorporated in a wide variety of ways. Types of one or more secondantigens (or additional antigens that are not coupled to the populationof synthetic nanocarriers) suitable for use with the present inventionhave been discussed elsewhere herein.

There are a wide variety of ways of incorporating the one or more firstor more second antigens (or additional antigens that are not coupled tothe population of synthetic nanocarriers) into the inventive dosageform. In an embodiment, the one or more second antigens may be admixedinto the dosage form together with the population of syntheticnanocarriers. For instance, in an embodiment, a vaccine that comprisesthe one or more second antigens may be admixed with the population ofsynthetic nanocarriers to form the inventive dosage forms. Inembodiments, the inventive synthetic nanocarriers can be incorporatedinto the inventive dosage forms together with one or more first antigenthat are different, similar or the same as the one or more secondantigens in a wide variety of ways, including but not limited to: withor without adjuvant, utilizing or not utilizing another deliveryvehicle, administered separately at a different time-point and/or at adifferent body location and/or by a different immunization route.

In embodiments, the populations of synthetic nanocarriers may becombined with the one or more second antigens (which may be incorporatedin a wide variety of ways) to form dosage forms according to the presentinvention. The one or more second antigens may be provided in solutionform, suspension form, powder form, etc., and may be provided as avaccine formulation. For instance, in an embodiment, the one or moresecond antigen may be provided in the form of a hapten-carrier protein,oligosaccharide, oligosaccharide complex, oligosaccharide-carrierprotein fusion, live attenuated, or recombinant virus vaccineformulation, and the population of synthetic nanocarriers admixed withthe hapten-carrier protein, oligosaccharide, oligosaccharide complex,oligosaccharide-carrier protein fusion, live attenuated, or recombinantvirus vaccine formulation, to form a multivalent vaccine dosage form (orincrease the valency of the hapten-carrier protein or live attenuatedvirus vaccine formulations). In embodiments, the one or more secondantigens can be comprised in a vaccine against Anthrax; Diphtheria,Tetanus and/or Pertussis; Haemophilus influenzae type B; Hepatitis B;Hepatitis A; Hepatitis C; Herpes zoster (shingles); Human Papillomavirus(HPV); Influenza; Japanese Encephalitis; Tick-borne Encephalitis;Measles, Mumps and/or Rubella; Meningococcal disease; Pneumococcaldisease; Polio; Rabies; Rotavirus; Typhoid; Varicella; Vaccinia(Smallpox); or Yellow Fever. In other embodiments, the one or moresecond antigens are comprised in a commercially available vaccine,including but not limited to, BIOTHRAX, DAPTACEL, INFANRIX, TRIPEDIA,TRIHIBIT, KINRIX, PEDIARIX, PENTACEL, PEDVAXHIB, ACTHIB, HIBERIX,COMVAX, HAVRIX, VAQTA, ENGERIX-B, RECOMBIVAX HB, TWINRIX, ZOSTAVAX,GARDASIL, CERVARIX, FLUARIX, FLUVIRIN, FLUZONE, FLULAVAL, AFLURIA,AGRIFLU, FLUMIST, JE-VAX, IXIARO, M-M-R II, PROQUAD, MENOMUNE, MENACTRA,MENVEO, PNEUMOVAX 23, PREVNAR, PCV13, IPOL, IMOVAX RABIES, RABAVERT,ROTATEQ, ROTARIX, DECAVAC, BOOSTRIX, ADACEL, TYPHIM VI, VIVOTIF BERNA,VARIVAX, ACAM2000 or YF-VAX.

In another embodiment, the population of synthetic nanocarriers may becombined with proteins taken from an infectious organism, such as humaninfluenza A virus HA protein, either in proteinaceous form or invirus-like particles, to form a multivalent vaccine dosage formaccording to the invention. In another embodiment, the population ofsynthetic nanocarriers may be added to another population of syntheticnanocarriers that comprise the one or more second antigens to form amultivalent synthetic nanocarrier vaccine dosage form. In otherembodiments, additional antigens beyond the one or more first and/orsecond antigens can be incorporated into the dosage form (throughadmixing, and other techniques disclosed herein or knownconventionally). In embodiments, the inventive compositions providedherein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or moredifferent antigens.

In embodiments, the one or more first antigens and/or one or more secondantigens comprise or are obtained or derived from a virus of a family ofviruses shown below in Table 1. In another embodiment, the one or morefirst antigens and/or one or more second antigens comprise or areobtained or derived from a virus of a species provided in Table 1. Instill another embodiment, the one or more first antigens and/or one ormore second antigens comprise or are obtained or derived from an antigenprovided in Table 1.

TABLE 1 Viral Infectious Agents Family Exemplary Species ExemplaryAntigens Adenoviridae adenovirus VI, VII, E1A, E3-19K, 52KPicornaviridae coxsackievirus, VP1 hepatitis A virus Surface antigenpoliovirus, 3A protein, capsid protein Rhinovirus (e.g., nucleocapsid,surface projection, type 16) and transmembrane proteins HerpesviridaeHerpes simplex Capsid proteins (e.g., UL6, (type 1 and type 2) UL18,UL35, UL38, and UL19) Varicella-zoster Early antigen virus Epstein-barrvirus, Early antigen, capsid antigen Human Pp65, gB, p52 cytomegalovirusHuman herpesvirus, Latent nuclear antigen-1 (e.g., type 8)Hepadnaviridae Hepatitis B virus surface antigen Flaviviridae HepatitisC virus, NS3, Envelop protein (e.g., E2 yellow fever virus, domain)dengue virus, West Nile virus Retroviridae HIV gp120, p24, andlipopeptides Gag (17-35), Gag (253-284), Nef (66-97), Nef (116-145), andPol (325-355); see Roberts et al., J. Immunol. Methods, 365(1-2):27-37,2011 Orthomyxoviridae Influenza virus neuraminidase, surface antigen,Paramyxoviridae Measles virus, Nucleocapsid protein, matrix Mumps virusprotein, phosphoprotein, Parainfluenza virus fusion protein,hemagglutinin, Respiratory hemagglutinin-neuraminidase, syncytial virusglycoprotein, Human metapneumovirus Papillomaviridae Human E6, E7,capsid antigen papillomavirus (e.g., type 16 and 18) RhabdoviridaeRabies virus Envelope lipoprotein Togaviridae Rubella virus Capsidprotein Paroviridae Human bocarivus, Capsid protein, non-structuralParvovirus B19 protein (NS)

In embodiments, the one or more first antigens and/or one or more secondantigens comprise or are obtained or derived from a bacteria of a generaof bacteria shown below in Table 2. In another embodiment, the one ormore first antigens and/or one or more second antigens comprise or areobtained or derived from a bacterial species provided in Table 2. Instill another embodiment, the one or more first antigens and/or one ormore second antigens comprise or are obtained or derived from an antigenprovided in Table 2.

TABLE 2 Bacterial Infectious Agents Pathogenic Bacterial GeneraExemplary Species Exemplary Antigens Bordetella Bordetella pertussispertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN),and fimbriae (FIM 2/3) Borrelia Borrelia burgdorferi VlsE; DbpA and OspABrucella Brucella abortus Hia, PrpA, M1tA, L7/L12, D15, 0187, VirJ, Mdh,AfuA Brucella canis L7/L12 Brucella melitensis Out membrane proteinssuch as Omp28 Brucella suis Campylobacter Campylobacter jejuni; LPS, an100-kD antigen Chlamydia and Chlamydia pneumoniae See Richard et al., J.Infectious Chlamydophila Chlamydia trachomatis Diseases. 181:S521 (2000)Chlamydophila psittaci Clostridium Clostridium botulinum antigen typesA, B, C, D, and E Clostridium difficile F1iC, F1iD, and Cwp84Clostridium perfringens alpha-toxin, theta-toxin, fructose 1,6-biphosphate-aldolase (FBA), glyceraldehydes-3-phosphate dehydrogenase(GPD), pyruvate:ferredoxin oxidoreductase (PFOR), elongation factor-G(EF-G), and a hypothetical protein (HP) Clostridium tetani T toxinCorynebacterium Corynebacterium diphtheriae Toxoid antigen EnterococcusEnterococcus faecalis capsular polysaccharides Enterococcus faeciumEscherichia Escherichia coli See Moriel et al., PNAS 107(20):9072- 9077(2010) Francisella Francisella tularensis See Havlasova et al.,Proteomics 2(7):857-867, 2002 Haemophilus Haemophilus influenzaecapsular polysaccharides, Protein D, Helicobacter Helicobacter pyloriSee Bumann et al., Proteomics 4(10):2843-2843, 2004 LegionellaLegionella pneumophila Mip Leptospira Leptospira interrogans See Brownet al., Infect Immu 59(5):1772-1777, 1991 Listeria Listeriamonocytogenes nucleoprotein (NP) Mycobacterium* Mycobacterium lepraeMycobacterium tuberculosis RD1, PE35, PPE68, EsxA, EsxB, RD9, and EsxVMycobacterium ulcerans Mycoplasma Mycoplasma pneumoniae Hsp70 NeisseriaNeisseria gonorrhoeae Neisseria meningitidis See Litt et al., J.Infectious Disease 190(8):1488-1497, 2004 Pseudomonas Pseudomonasaeruginosa Lipopolysaccharides Rickettsia Rickettsia rickettsii Surfaceantigen Salmonella Salmonella typhi Salmonella typhimurium ShigellaShigella sonnei Staphylococcus Staphylococcus aureus See Vytvtska etal., Proteomics 2(5):580-590, 2002; Etz et al., PNAS 99(10):6573-6578;2002 Staphylococcus epidermidis Staphylococcus saprophyticusStreptococcus Streptococcus agalactiae Streptococcus pneumoniae Sp 1,Sp2, Sp3 Streptococcus pyogenes Lei et al., J. Infectious Disease189(1):79-89, 2004 Treponema Treponema pallidum GlycerophosphodiesterPhosphodiesterase Vibrio Vibrio cholerae Outer membrane proteins such asOmpK Yersinia Yersinia pestis Chaperone-usher protein, capsular protein(F1), and V protein

In other embodiments, the one or more first antigens and/or one or moresecond antigens comprise or are obtained or derived from a fungus of agenera of fungi shown below in Table 3. In another embodiment, the oneor more first antigens and/or one or more second antigens comprise orare obtained or derived from a fungal species provided in Table 3. Instill another embodiment, the one or more first antigens and/or one ormore second antigens comprise or are obtained or derived from an antigenprovided in Table 3.

TABLE 3 Fungal Infectious Agents Genera Exemplary Species ExemplaryAntigens Candida C. albicans Surface antigens, see also Thomas et al.,Proteomics 6(22):6033-6041, 2006 Aspergillus Aspergillus fumigatusStevens et al., Medical Mycology 49 and Aspergillus (Suppl.1):5170-5176, 2011 flavus. Cryptococcus Cryptococcus Capsularglycoproteins, neoformans, Cryptococcus laurentii and Cryptococcusalbidus, Cryptococcus gattii Histoplasma Histoplasma Yps3P, Hsp60capsulatum Pneumocystis Pneumocystis Major surface proteins (Msg) suchas jirovecii MsgC1, MsgC3, MsgC8, and MsgC9 Stachybotrys StachybotrysSchS34, chartarum

Combination of the population of synthetic nanocarriers and the one ormore second antigens may be accomplished using traditionalpharmaceutical mixing methods. These include liquid-liquid mixing inwhich two or more suspensions, containing a population of syntheticnanocarrier or the one or more second antigens, are directly combined orare brought together via one or more vessels containing diluent. Assynthetic nanocarriers may also be produced or stored in a powder form,dry powder-powder mixing could be performed if the one or more secondantigens are available in powder, as could the re-suspension of two ormore powders in a common media. Depending on the properties and theinteraction potential of the synthetic nanocarriers and the one or moresecond antigens, there may be advantages conferred to one or anotherroute of mixing. Techniques suitable for use in practicing the presentinvention may be found in Handbook of Industrial Mixing: Science andPractice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and SuzanneM. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Scienceof Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, ChurchillLivingstone. In an embodiment, inventive synthetic nanocarriers aresuspended in sterile saline solution for injection together with apreservative.

Doses of dosage forms contain varying amounts of populations ofsynthetic nanocarriers and varying amounts of one or more secondantigens, according to the invention. The amount of syntheticnanocarriers and/or one or more second antigens present in the inventivedosage forms can be varied according to the nature of the antigens, thetherapeutic benefit to be accomplished, and other such parameters. Inembodiments, dose ranging studies can be conducted to establish optimaltherapeutic amount of the population of synthetic nanocarriers and theamount of one or more second antigens to be present in the dosage form.In embodiments, the population of synthetic nanocarriers and the one ormore second antigens are present in the dosage form in an amounteffective to generate an immune response to the one or more firstantigens and the one or more second antigens upon administration to asubject. It may be possible to determine amounts of the first, second,and/or subsequent antigens effective to generate an immune responseusing conventional dose ranging studies and techniques in subjects.

In embodiments, the inventive dosage forms can be formulated by admixinguncoupled adjuvants in the same vehicle or delivery system as thepopulation of synthetic nanocarriers and the one or more secondantigens. Such adjuvants may include, but are not limited to mineralsalts, such as alum, alum combined with monphosphoryl lipid (MPL) A ofEnterobacteria, such as Escherihia coli, Salmonella minnesota,Salmonella typhimurium, or Shigella flexneri or specifically with MPL®(AS04), MPL A of above-mentioned bacteria separately, saponins, such asQS-21, Quil-A, ISCOMs, ISCOMATRIX™, emulsions such as MF59™, Montanide®ISA 51 and ISA 720, AS02 (QS21+squalene+MPL®), AS15, liposomes andliposomal formulations such as AS01, synthesized or specificallyprepared microparticles and microcarriers such as bacteria-derived outermembrane vesicles (OMV) of N. gonorrheae, Chlamydia trachomatis andothers, or chitosan particles, depot-forming agents, such as Pluronic®block co-polymers, specifically modified or prepared peptides, such asmuramyl dipeptide, aminoalkyl glucosaminide 4-phosphates, such as RC529,or proteins, such as bacterial toxoids or toxin fragments. The doses ofsuch other adjuvants can be determined using conventional dose rangingstudies. In embodiments, adjuvant that is not coupled to the recitedpopulation synthetic nanocarriers may be the same or different fromadjuvant that is coupled to the synthetic nanocarriers.

Typical inventive compositions that comprise synthetic nanocarriers maycomprise inorganic or organic buffers (e.g., sodium or potassium saltsof phosphate, carbonate, acetate, or citrate) and pH adjustment agents(e.g., hydrochloric acid, sodium or potassium hydroxide, salts ofcitrate or acetate, amino acids and their salts) antioxidants (e.g.,ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20,polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate),solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol,trehalose), osmotic adjustment agents (e.g., salts or sugars),antibacterial agents (e.g., benzoic acid, phenol, gentamicin),antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g.,thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers andviscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488,carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethyleneglycol, ethanol).

Compositions according to the invention comprise inventive syntheticnanocarriers in combination with pharmaceutically acceptable excipients.The compositions may be made using conventional pharmaceuticalmanufacturing and compounding techniques to arrive at useful dosageforms. Techniques suitable for use in practicing the present inventionmay be found in Handbook of Industrial Mixing: Science and Practice,Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta,2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of DosageForm Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone.In an embodiment, inventive synthetic nanocarriers are suspended insterile saline solution for injection together with a preservative.

It is to be understood that the compositions of the invention can bemade in any suitable manner, and the invention is in no way limited tocompositions that can be produced using the methods described herein.Selection of an appropriate method may require attention to theproperties of the particular moieties being associated.

In some embodiments, inventive synthetic nanocarriers are manufacturedunder sterile conditions or are terminally sterilized. This can ensurethat resulting composition are sterile and non-infectious, thusimproving safety when compared to non-sterile compositions. Thisprovides a valuable safety measure, especially when subjects receivingsynthetic nanocarriers have immune defects, are suffering frominfection, and/or are susceptible to infection. In some embodiments,inventive synthetic nanocarriers may be lyophilized and stored insuspension or as lyophilized powder depending on the formulationstrategy for extended periods without losing activity.

The inventive compositions may be administered by a variety of routes ofadministration, including but not limited to subcutaneous,intramuscular, intradermal, oral, intranasal, transmucosal, sublingual,rectal, ophthalmic, transdermal, transcutaneous or by a combination ofthese routes.

Doses of dosage forms contain varying amounts of synthetic nanocarriersor populations thereof and varying amounts of antigens and/or adjuvants,according to the invention. The amount of synthetic nanocarriers and/orantigens and/or adjuvants present in the inventive dosage forms can bevaried according to the nature of the antigens, the therapeutic benefitto be accomplished, and other such parameters. In embodiments, doseranging studies can be conducted to establish optimal therapeutic amountof the synthetic nanocarriers or population thereof and the amount ofantigens and/or adjuvant to be present in the dosage form. Inembodiments, the synthetic nanocarriers and the antigens and/oradjuvants are present in the dosage form in an amount effective togenerate an immune response to the antigens upon administration to asubject. It may be possible to determine amounts of the antigens and/oradjuvants effective to generate an immune response using conventionaldose ranging studies and techniques in subjects. Inventive dosage formsmay be administered at a variety of frequencies. In a preferredembodiment, at least one administration of the dosage form is sufficientto generate a pharmacologically relevant response. In more preferredembodiment, at least two administrations, at least threeadministrations, or at least four administrations, of the dosage formare utilized to ensure a pharmacologically relevant response.

The compositions and methods described herein can be used to induce,enhance, suppress, modulate, direct, or redirect an immune response. Thecompositions and methods described herein can be used in the diagnosis,prophylaxis and/or treatment of conditions such as cancers, infectiousdiseases, metabolic diseases, degenerative diseases, autoimmunediseases, inflammatory diseases, immunological diseases, or otherdisorders and/or conditions. The compositions and methods describedherein can also be used for the prophylaxis or treatment of anaddiction, such as an addiction to nicotine or a narcotic. Thecompositions and methods described herein can also be used for theprophylaxis and/or treatment of a condition resulting from the exposureto a toxin, hazardous substance, environmental toxin, or other harmfulagent.

The subjects provided herein can have or be at risk of having cancer.Cancers include, but are not limited to, breast cancer; biliary tractcancer; bladder cancer; brain cancer including glioblastomas andmedulloblastomas; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; hematologicalneoplasms including acute lymphocytic and myelogenous leukemia, e.g., BCell CLL; T-cell acute lymphoblastic leukemia/lymphoma; hairy cellleukemia; chronic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia/lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; neuroblastomas; oral cancer including squamouscell carcinoma; ovarian cancer including those arising from epithelialcells, stromal cells, germ cells and mesenchymal cells; pancreaticcancer; prostate cancer; rectal cancer; sarcomas includingleiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, andosteosarcoma; skin cancer including melanoma, Merkel cell carcinoma,Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor.

The subjects provided herein can have or be at risk of having aninfection or infectious disease. Infections or infectious diseasesinclude, but are not limited to, viral infectious diseases, such asAIDS, Chickenpox (Varicella), Common cold, Cytomegalovirus Infection,Colorado tick fever, Dengue fever, Ebola hemorrhagic fever, Hand, footand mouth disease, Hepatitis, Herpes simplex, Herpes zoster, HPV,Influenza (Flu), Lassa fever, Measles, Marburg hemorrhagic fever,Infectious mononucleosis, Mumps, Norovirus, Poliomyelitis, Progressivemultifocal leukencephalopathy, Rabies, Rubella, SARS, Smallpox(Variola), Viral encephalitis, Viral gastroenteritis, Viral meningitis,Viral pneumonia, West Nile disease and Yellow fever; bacterialinfectious diseases, such as Anthrax, Bacterial Meningitis, Botulism,Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis(Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever,Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever,Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia,Typhoid Fever, Typhus and Urinary Tract Infections; parasitic infectiousdiseases, such as African trypanosomiasis, Amebiasis, Ascariasis,Babesiosis, Chagas Disease, Clonorchiasis, Cryptosporidiosis,Cysticercosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis,Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Free-livingamebic infection, Giardiasis, Gnathostomiasis, Hymenolepiasis,Isosporiasis, Kalaazar, Leishmaniasis, Malaria, Metagonimiasis, Myiasis,Onchocerciasis, Pediculosis, Pinworm Infection, Scabies,Schistosomiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinellosis,Trichinosis, Trichuriasis, Trichomoniasis and Trypanosomiasis; fungalinfectious disease, such as Aspergillosis, Blastomycosis, Candidiasis,Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis(Athlete's Foot) and Tinea cruris; prion infectious diseases, such asAlpers' disease, Fatal Familial Insomnia, Gerstmann-Sträussler-Scheinkersyndrome, Kuru and Variant Creutzfeldt-Jakob disease.

EXAMPLES Example 1 Synthetic Nanocarriers with Covalently CoupledAdjuvant

Nanocarriers comprising PLGA-R848, PLA-PEG-N3, and ova peptide wereprepared via double emulsion method wherein the ova peptide wasencapsulated in the nanocarriers.

The polyvinyl alcohol (Mw=11 KD-31 KD, 87-89% partially hydrolyzed) waspurchased from JT Baker. Ovalbumin peptide 323-339 was obtained fromBachem Americas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Part #4065609). PLGA-R848, and PLA-PEG-N3 conjugates were synthesized andpurified.

The above materials were used to prepare the following solutions:

1. PLGA-R848 conjugate in methylene chloride @ 100 mg/mL

2. PLA-PEG-N3 in methylene chloride @ 100 mg/mL

3. Ovalbumin peptide 323-339 in 0.13N HCl @ 70 mg/mL

4. Polyvinyl alcohol in 100 mM pH 8 phosphate buffer @50 mg/mL

Solution #1 (0.75 mL) and solution #2 (0.25 mL) were combined andsolution #3 (0.1 mL) or 0.13N HCl (0.1 mL) was added in a small vesseland the mixture was sonicated at 50% amplitude for 40 seconds using aBranson Digital Sonifier 250. To this emulsion was added solution # 4(2.0 mL) and sonication at 30% amplitude for 40 seconds using theBranson Digital Sonifier 250 formed the second emulsion. This was addedto a stiffing beaker containing a 70 mM pH 8 phosphate buffer solution(30 mL), and this mixture was stirred at room temperature for 2 hours toform the nanocarriers.

To wash the nanocarriers, a portion of the nanoparticle dispersion (26.5mL) was transferred to a 50 mL centrifuge tube and spun at 9500 rpm(13,800 g) for one hour at 4° C., the supernatant was removed, and thepellet was re-suspended in 26.5 mL of phosphate buffered saline. Thecentrifuge procedure was repeated and the pellet was re-suspended in 8.3g of phosphate buffered saline for a final nanocarrier dispersion ofabout 10 mg/mL.

To a suspension of the synthetic nanocarriers (10 mg/mL in PBS (pH 7.4buffer), 5 mL, containing about 12.5 mg (MW: 20,000; 0.000625 mmol) ofPLA-PEG-N3) was added L2 derived peptideH-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-Ile-Pro-Lys-Val-X(SEQ ID NO:2); wherein X is a linker group comprising an acetylenelinker (33 mg) with gentle stiffing. A solution of sodium ascorbate (100mM in H2O, 0.3 mL) was added, followed by CuSO4 solution (10 mM inwater, 0.6 mL). The resulting light yellow suspension was stirred at 20C for 15 h and additional CuSO4 solution (0.3 mL) and sodium ascorbatesolution (0.15 mL) were added. The suspension was stirred for 5 h at 20C and diluted with PBS buffer (pH 7.4) to 10 mL and centrifuged toremove the supernatant. The residual nanocarrier pellets were washedtwice with PBS buffer. The washed nanocarriers were then re-suspended in5 mL of PBS buffer and stored frozen. The conjugation of L2 peptide onthe surface of the synthetic nanocarriers was confirmed by HPLC analysisof the digested nanocarriers and by bioassay.

Example 2 Composition with Synthetic Nanocarriers and Uncoupled Antigen(Prophetic)

A 4 mL portion of the synthetic nanocarrier suspension from Example 1containing 8 mg of L2 substituted nanocarriers is centrifuged to settlethe particles. The supernatant is discarded and a 0.5-mL suspension ofGardasil®, Human Papillomavirus Quadrivalent (Types 6, 11, 16, and 18)Vaccine containing purified virus-like particles (VLPs) of the majorcapsid (L1) protein of HPV Types 6, 11, 16, and 18 is added. Thecombination vaccine is agitated to re-suspend the nanocarriers and theresulting suspension is stored at −20° C. prior to use.

Example 3 Synthetic Nanocarriers with Non-Covalently Coupled Adjuvant[Prophetic]

DNA containing cationic disulfide PRINT nanocarriers are produced by themethod described in the patent application of DeSimone, WO2008118861,example 16 with the exception that the ssDNA-fluorescein of example 16is replaced by the phosphorothioated DNA CpG 7909. After isolation, thecationic nanocarriers are suspended in 1.0 mL of PBS solution containing10 mg/mL of heparin. After stirring at room temperature for 2 hours, thenanocarriers are isolated by centrifugation and are washed twice withPBS by centrifugation and decantation. The nanocarriers containing CpG7909 with surface adsorbed heparin are re-suspended in 1.0 mL of PBS andare stored at −20° C. prior to use.

Example 4 Composition with Synthetic Nanocarriers and Uncoupled Antigen(Prophetic)

A 1 mL portion of the synthetic nanocarrier suspension from Example 3containing 10 mg of heparin substituted nanocarriers is centrifuged tosettle the particles. The supernatant is discarded and a 1-mL suspensionof Recombivax HB® or Engerix-B®, human hepatitis B Virus (HBV) vaccinescontaining purified proteinaceous particles consisting of the majorsurface antigen (HBsAg) protein of HBV is added. The combination vaccineis agitated to re-suspend the nanocarriers and the resulting suspensionis stored at −20° C. prior to use. A similar process is used to combinethe heparin-substituted nanocarriers of Example 3 with a 1 mL suspensionof bivalent vaccine against human hepatitis A and B viruses (Twinrix®),consisting of purified HBsAg and inactivated human hepatitis A virus.

Example 5 Synthetic Nanocarriers with Covalently Coupled Adjuvant[Prophetic] Example 5A Preparation of R848 Covalently Attached to aThiol

3,3′-dithio bis-propionic acid (cat #109010) is purchased from AldrichChemical Company. R848 is synthesized at Selecta Biosciences. A solutionof 3,3′-dithio bis-propionic acid (2.10 gm, 1.0×10⁻² moles) and HBTU(15.2 g, 4×10⁻² moles) in EtOAc (450 mL) is stirred at room temperatureunder argon for 45 min. Compound R848 (6.28 g, 2×10⁻² moles)) is added,followed by DIPEA (20.9 mL, 1.2×10⁻¹ moles). The mixture is stirred atroom temperature for 6 h and then at 50-55° C. for 15 h. After cooling,the mixture is washed with 1% citric acid solution (2×40 mL), water (40mL) and brine solution (40 mL). The solution is dried over Na₂SO₄ (10 g)and, after filtration, the ethyl acetate is removed under vacuum. Theproduct is recrystallized from 2-methoxyethanol to provide 6.5 gm (78%)of a white solid product.

The disulfide from above (5.0 gm) is dissolved in chloroform (200 mL)and the solution is treated with dithiothreitol (1.0 gm). After stiffingat room temperature for 2 hours, the chloroform solution is washed withwater (100 mL) and is then dried over sodium sulfate. After filtrationto remove the drying agent, the chloroform is removed under vacuum andthe solid remaining is purified by chromatography on silica using 10%methanol in methylene chloride as eluent. The fractions containing thethiol-R848 conjugate are pooled and evaporated to give 3.5 gm (70%) ofthe thiol-R848 conjugate as a white solid.

Example 5B Preparation of Nanocarriers

Gold synthetic nanocarriers are prepared as described in example (a) ofUS patent application 2009 0104268 A1 to Midatech Limited except thatpeptide BC11 is replaced with the thiol R848 conjugate from Example 5Aabove and the oligosaccharide antigens are replaced with L2 derivedpeptideH-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-Ile-Pro-Lys-Val-X(SEQ ID NO:2); wherein X is a linker group comprising a cysteineresidue. After washing and concentration as described in the Midatechapplication, the particles weighing 1.0 mg are used as described inExample 6.

Example 6 Composition with Synthetic Nanocarriers and Uncoupled Antigen(Prophetic)

A 1.0 mg portion of the gold nanocarriers from Example 5 are added to a1 mL of oral suspension of live recombinant anti-rotaviral vaccineRotarix® against gastroenteritis induced by type G1 and non-G1 (G3, G4,and G9) rotavirus types. The combination oral vaccine is agitated tore-suspend the nanocarriers and the resulting suspension is stored at−20° C. prior to use as a combination oral vaccine.

Example 7 Synthetic Nanocarriers with T-helper Antigen and Adjuvant

Ovalbumin peptide 323-339 amide acetate salt, was purchased from BachemAmericas Inc. (3132 Kashiwa Street, Torrance Calif. 90505. Product code4065609.) PLGA-R848, poly-D/L-lactide-co-glycolide,4-amino-2-(ethoxymethyl)-α,α-dimethyl-1H-imidazo[4,5-c]quinoline-1-ethanolamide of approximately 7,000 Da made from PLGA of 3:1 lactide toglycolide ratio and having approximately 8.5% w/w conjugated resiquimodcontent was custom manufactured at Princeton Global Synthesis (300George Patterson Drive #206, Bristol, Pa. 19007.) PLA-PEG-C6-N3, blockco-polymer consisting of a poly-D/L-lactide (PLA) block of approximately23000 Da and a polyethylene glycol (PEG) block of approximately 2000 Dathat is terminated by an amide-conjugated C6H12 linker to an azide, wassynthesized by conjugating HO-PEG-COOH to an amino-C6H12-azide and thengenerating the PLA block by ring-opening polymerization of the resultingHO-PEG-C6-N3 with dl-lactide. Polyvinyl alcohol PhEur, USP (85-89%hydrolyzed, viscosity of 3.4-4.6 mPa·s) was purchased from EMD ChemicalsInc. (480 South Democrat Road Gibbstown, N.J. 08027. Part Number 4-88).

Solutions were prepared as follows:

Solution 1: Ovalbumin peptide 323-339 @ 20 mg/mL was prepared in 0.13NHCl at room temperature.

Solution 2: PLGA-R848 @ 50 mg/mL and PLA-PEG-C6-N3 @ 50 mg/mL indichloromethane was prepared by dissolving each separately at 100 mg/mLin dichloromethane then combining in equal parts by volume.

Solution 3: Polyvinyl alcohol @ 50 mg/mL in 100 mM in 100 mM phosphatebuffer, pH 8

Solution 4: 70 mM phosphate buffer, pH 8

A primary (W1/O) emulsion was first created using Solution 1 andSolution 2. Solution 1 (0.2 mL) and Solution 2 (1.0 mL) were combined ina small glass pressure tube and sonicated at 50% amplitude for 40seconds using a Branson Digital Sonifier 250. A secondary (W1/O/W2)emulsion was then formed by adding Solution 3 (2.0 mL) to the primaryemulsion, vortexing to create a course dispersion, and then sonicatingat 30% amplitude for 40 seconds using the Branson Digital Sonifier 250.

The secondary emulsion was added to an open 50 mL beaker containing 70mM phosphate buffer solution (30 mL) and stirred at room temperature for2 hours to allow the dichloromethane to evaporate and the nanocarriersto form in suspension. A portion of the suspended nanocarriers waswashed by transferring the nanocarrier suspension to a centrifuge tube,spinning at 21,000 rcf for 45 minutes, removing the supernatant, andre-suspending the pellet in phosphate buffered saline. This washingprocedure was repeated, and then the pellet was re-suspended inphosphate buffered saline to achieve a nanocarrier suspension having anominal concentration of 10 mg/mL on a polymer basis. Two identicalbatches were created and then combined to form a single homogenoussuspension at which was stored frozen at −20° C. until further use.

TABLE 4 Nanocarrier Characterization Effective TLR Agonist, NanocarrierDiameter (nm) % w/w Antigen, % w/w 209 R848, 4.2 Ova 323-339 peptide,2.4

Example 8 Immunization with Synthetic Nanocarriers with Coupled Antigenand Free Protein Without Admixed Adjuvant Materials and Methods

(1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 andOva-peptide, prepared as above in Example 7, 7 mg/mL suspension in PBS.

(2) M2e peptide modified with an alkyne linker attached to C-terminalGly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW 2650, TFA salt;Sequence:

(SEQ ID NO: 3) H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DIwater; sodium ascorbate, 200 mM in DI water freshly prepared.

(4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifuge. A solution of M2e peptide (20 mg) in 2 mL PBS buffer wasadded. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mL ofTHPTA ligand (200 mM) was added, followed by 0.4 mL of sodium ascorbate(200 mM). The resulting light yellow suspension was stirred in dark atambient room temperature for 18 h. The suspension was then diluted withPBS buffer to 10 mL and centrifuged to remove the supernatant. TheNC-M2e conjugates were further pellet washed twice with 10 mL PBS bufferand resuspended in pH 7.4 buffer at final concentration of ca. 6 mg/mL(ca. 4 mL) and stored at 4° C.

Results

Antibody titers in mice immunized with a combination of NC-M2e and freehemagglutinin from H5N1 avian influenza strain (Vietnam) were measured.NC-M2e contained OP-II T-helper peptide (2.4%) and R848 adjuvant (4.2%).Each bar represents the titer against antigen. Five animals per groupwere immunized s.c. with 120 μg of NC and 10 μg of H5 hemaggutinin perinjection, 2 times with 3-wk intervals. Titers for day 33 after thefirst immunization are shown (ELISA against PLA-PEG-M2e and H5hemagglutinin, respectively).

The results show that immunization with a combination of NC carrying anantigen admixed to a free protein without an admixed adjuvant results ingeneration of antibodies to both NC-carried antigen and to a freeprotein. When a NC containing surface M2e peptide from influenza A virus(ectodomain of M2 matrix protein, amino acids 2-27) was admixed to freeinfluenza A virus hemagglutinin protein and used for animalimmunization, a strong humoral response was induced in all animalsagainst both M2e peptide and hemagglutinin (FIG. 1). No reactivity wasdetected in the sera of preimmune mice.

Example 9 Immunization with Synthetic Nanocarriers with Coupled Antigenand Free Protein with Admixed Adjuvant Materials and Methods

(1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 andOva-peptide, prepared as above in Example 7, 7 mg/mL suspension in PBS.

(2) M2e peptide modified with an alkyne linker attached to C-terminalGly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW 2650, TFA salt;Sequence:

(SEQ ID NO: 3) H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH.

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DIwater; sodium ascorbate, 200 mM in DI water freshly prepared.

(4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifuge. A solution of M2e peptide (20 mg) in 2 mL PBS buffer wasadded. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mL ofTHPTA ligand (200 mM) was added, followed by 0.4 mL of sodium ascorbate(200 mM). The resulting light yellow suspension was stirred in dark atambient room temperature for 18 h. The suspension was then diluted withPBS buffer to 10 mL and centrifuged to remove the supernatant. TheNC-M2e conjugates were further pellet washed twice with 10 mL PBS bufferand resuspended in pH 7.4 buffer at final concentration of ca. 6 mg/mL(ca. 4 mL) and stored at 4° C.

Results

Antibody titers in mice immunized with a combination of NC-M2e and freehemagglutinin from H5N1 avian influenza strain (Vietnam) admixed with 80μg of alum. NC-M2e contained OP-II T-helper peptide (2.4%) and R848adjuvant (4.2%). Each bar represents the titer against antigen. Fiveanimals per group were immunized s.c. with 120 μg of NC and 10 μg of H5hemaggutinin per injection, 2 times with 3-wk intervals. Titers for day33 after the first immunization are shown (ELISA against PLA-PEG-M2e andH5 hemagglutinin, respectively).

The results show that immunization with a combination of a NC carryingan antigen admixed to a second antigen (free protein) with an admixedadjuvant results in generation of antibodies to both NC-carried antigenand to the second antigen. When a NC containing surface M2e peptide frominfluenza A virus (ectodomain of M2 matrix protein, amino acids 2-27)was admixed to free influenza A virus hemagglutinin protein and used foranimal immunization admixed to alum (Imject Alum, Pierce), a stronghumoral response was induced in all animals against both M2e peptide andhemagglutinin (FIG. 2). No reactivity was detected in the sera ofpreimmune mice.

Example 10 Immunization with Synthetic Nanocarriers with Coupled Antigenand Virus Vaccine and Adjuvant Materials and Methods

(1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 andOva-peptide, prepared as above in Example 7, 7 mg/mL suspension in PBS.

(2) M2e peptide modified with an alkyne linker attached to C-terminalGly; CS Bio Co, Catalog No. CS4956, Lot: H308, MW 2650, TFA salt;Sequence:

(SEQ ID NO: 3) H-Met-Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Thr-Arg-Asn-Glu-Trp-Glu-Cys-Arg-Cys-Ser-Asp-Gly-Gly- NHCH2CCH.

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DIwater; sodium ascorbate, 200 mM in DI water freshly prepared.

(4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifuge. A solution of M2e peptide (20 mg) in 2 mL PBS buffer wasadded. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mL ofTHPTA ligand (200 mM) was added, followed by 0.4 mL of sodium ascorbate(200 mM). The resulting light yellow suspension was stirred in dark atambient room temperature for 18 h. The suspension was then diluted withPBS buffer to 10 mL and centrifuged to remove the supernatant. TheNC-M2e conjugates were further pellet washed twice with 10 mL PBS bufferand resuspended in pH 7.4 buffer at final concentration of ca. 6 mg/mL(ca. 4 mL) and stored at 4° C.

Results

Antibody titers in mice immunized with a combination of NC-M2e andbeta-propiolactone-inactivated influenza A virus H1N1 (H1N1 NewCaledonia/20/99/IVR 116) admixed with 80 μg of alum were measured.NC-M2e contained OP-II T-helper peptide (2.4%) and R848 adjuvant (4.2%).Each bar represents the titer against antigen. Five animals per groupwere immunized s.c. with 120 μg of NC and 1 μg of inactivated,thimerosal-containing H1N1 New Caledonia per injection, 2 times with3-wk intervals. Titers for day 33 after the first immunization are shown(ELISA against PLA-PEG-M2e and H1N1 New Caledonia, respectively).

The results show that immunization with a combination of NC carrying anantigen admixed with an inactivated virus vaccine and an adjuvantresults in generation of antibodies to both NC-carried antigen and aninactivated virus. When a NC containing surface M2e peptide frominfluenza A virus (ectodomain of M2 matrix protein, amino acids 2-27)was admixed to inactivated influenza A virus H1N1 and used for animalimmunization admixed to alum (Imject Alum, Pierce), a strong humoralresponse was induced in all animals against both M2e peptide andinactivated influenza A virus H1N1 (FIG. 3). No reactivity was detectedin the sera of preimmune mice.

Example 11 Immunization with Synthetic Nanocarriers with Coupled Antigenand Recombinant Vaccine with Adjuvant Materials and Methods

(1) Nanocarriers with surface PEG-C6-N3 containing PLGA-R848 andOva-peptide, prepared as above in Example 7, 7 mg/mL suspension in PBS.

(2) HPV16 L2 peptide modified with an alkyne linker attached toC-terminal Lys amino group; Bachem Americas, Inc, Lot B06055, MW 2595,TFA salt; Sequence:

(SEQ ID NO: 2) H-Ala-Thr-Gln-Leu-Tyr-Lys-Thr-Cys-Lys-Gln-Ala-Gly-Thr-Cys-Pro-Pro-Asp-Ile-Ile-Pro-Lys-Val- Lys(5-hexynoy1)-NH2(with Cys-Cys disulfide bond).

(3) Catalysts: CuSO4, 100 mM in DI water; THPTA ligand, 200 mM in DIwater; sodium ascorbate, 200 mM in DI water freshly prepared.

(4) pH 7.4 PBS buffer.

The NC suspension (7 mg/mL, 4 mL) was concentrated to ca. 1 mL in volumeby centrifuge. A solution of L2 peptide (20 mg) in 2 mL PBS buffer wasadded. A pre-mixed solution of 0.2 mL of CuSO4 (100 mM) and 0.2 mL ofTHPTA ligand (200 mM) was added, followed by 0.4 mL of sodium ascorbate(200 mM). The resulting light yellow suspension was stirred in dark atambient room temperature for 18 h. The suspension was then diluted withPBS buffer to 10 mL and centrifuged to remove the supernatant. The NC-L2conjugates were further pellet washed twice with 10 mL PBS buffer andresuspended in pH 7.4 buffer at final concentration of ca. 6 mg/mL (ca.4 mL) and stored at 4° C.

Results

Antibody titers in mice immunized with a combination of NC-L2-peptideand HBsAg strain ayw produced in the yeast Saccharomyces cerevisiaeadmixed with 80 μg of alum were measured. NC-L2-peptide contained OP-IIT-helper peptide (2.4%) and R848 adjuvant (4.2%). Each bar representsthe titer against antigen indicated. Five animals per group wereimmunized s.c. with 120 μg of NC and 0.6 μg of recombinant HBsAg, perinjection, 2 times with 3-wk intervals. Titers for day 33 after thefirst immunization are shown (ELISA against PLA-PEG-L2 and HBsAg ayw,respectively).

The results show that immunization with a combination of a NC carryingan antigen admixed to a recombinant vaccine and an adjuvant results ingeneration of antibodies to both NC-carried antigen and to aninactivated virus. When a NC containing surface L2 peptide from HPV-16virus minor capsid L2 protein (amino acids 17-36) was admixed torecombinant hepatitis B surface antigen (HBsAg) and used for animalimmunization admixed to alum (Imject Alum, Pierce), a strong humoralresponse was induced in all animals against both L2 peptide andrecombinant HBsAg (FIG. 4). No reactivity was detected in the sera ofpreimmune mice.

1. A dosage form comprising: (1) a first population of syntheticnanocarriers that have one or more first antigens coupled to them, (2)one or more second antigens that are not coupled to the syntheticnanocarriers, and (3) a pharmaceutically acceptable excipient.
 2. Thedosage form of claim 1, further comprising one or more adjuvants thatare coupled to the synthetic nanocarriers of the first population ofsynthetic nanocarriers.
 3. The dosage form of claim 2, wherein the oneor more coupled adjuvants comprise Pluronic® block co-polymers,specifically modified or prepared peptides, muramyl dipeptide,aminoalkyl glucosaminide 4-phosphates, RC529, bacterial toxoids, toxinfragments, agonists of Toll-Like Receptors 2, 3, 4, 5, 7, 8, 9 and/orcombinations thereof; adenine derivatives; immunostimulatory DNA;immunostimulatory RNA; imidazoquinoline amines, imidazopyridine amines,6,7-fused cycloalkylimidazopyridine amines, 1,2-bridged imidazoquinolineamines; imiquimod; resiquimod; type I interferons; poly I:C; bacteriallipopolysacccharide (LPS); VSV-G; HMGB-1; flagellin or portions orderivatives thereof; or immunostimulatory DNA molecules comprising CpGs.4. The dosage form of claim 2, wherein the one or more coupled adjuvantscomprise an agonist of Toll-Like Receptor 2, 3, 4, 7, 8 or
 9. 5. Thedosage form of claim 2, wherein the one or more coupled adjuvantscomprise an imidazoquinoline or oxoadenine.
 6. The dosage form of claim5, wherein the imidazoquinoline comprises resiquimod or imiquimod. 7-11.(canceled)
 12. The dosage form of claim 1, wherein the one or more firstantigens comprise a B cell antigen or a T cell antigen. 13-18.(canceled)
 19. The dosage form of claim 1, wherein the dosage formcomprises a vaccine that comprises the second antigen that is notcoupled to the synthetic nanocarriers.
 20. The dosage form of claim 19,wherein the vaccine comprises a hapten-carrier conjugate, a virus-likeparticle, a synthetic nanocarrier vaccine, a subunit protein vaccine, oran attenuated virus.
 21. The dosage form of claim 19, wherein thevaccine is against Anthrax; Diphtheria, Tetanus and/or Pertussis;Haemophilus influenzae type B; Hepatitis B; Hepatitis A; Hepatitis C;Herpes zoster (shingles); Human Papillomavirus (HPV); Influenza;Japanese Encephalitis; Tick-borne Encephalitis; Measles, Mumps and/orRubella; Meningococcal disease; Pneumococcal disease; Polio; Rabies;Rotavirus; Typhoid; Varicella; Vaccinia (Smallpox); or Yellow Fever. 22.(canceled)
 23. The dosage form of claim 1, wherein the one or more firstantigens and/or one or more second antigens are obtained or derived froma virus of the Adenoviridae, Picornaviridae, Herpesviridae,Hepadnaviridae, Flaviviridae, Retroviridae, Orthomyxoviridae,Paramyxoviridae, Papillomaviridae, Rhabdoviridae, Togaviridae orParoviridae family. 24-25. (canceled)
 26. The dosage form of claim 1,wherein the one or more first antigens and/or one or more secondantigens are obtained or derived from a bacteria of the Bordetella,Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila,Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella,Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema Vibrio orYersinia genus. 27-28. (canceled)
 29. The dosage form of claim 1,wherein the one or more first antigens and/or one or more secondantigens are obtained or derived from a fungus of the Candida,Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrysgenus. 30-31. (canceled)
 32. The dosage form of claim 1, wherein the oneor more first antigens and/or one or more second antigens are obtainedor derived from one or more proteins of human papilloma virus. 33-34.(canceled)
 35. The dosage form of claim 1, wherein the one or more firstantigens and/or one or more second antigens are obtained or derived fromone or more proteins of hepatitis B virus. 36-37. (canceled)
 38. Thedosage form of claim 35, wherein when the one or more first antigens areobtained or derived from hepatitis B virus, the one or more secondantigens are obtained or derived from one or more proteins of humanpapilloma virus.
 39. The dosage form of claim 35, wherein when the oneor more second antigens are obtained or derived from hepatitis B virus,the one or more first antigens are obtained or derived from one or moreproteins of human papilloma virus.
 40. (canceled)
 41. The dosage form ofclaim 1, wherein the one or more first antigens and/or one or moresecond antigens are obtained or derived from one or more proteins ofinfluenza virus. 42-47. (canceled)
 48. The dosage form of claim 1,wherein the first synthetic nanocarriers comprise lipid-basednanoparticles, polymeric nanoparticles, metallic nanoparticles,surfactant-based emulsions, dendrimers, buckyballs, nanowires,virus-like particles, peptide or protein-based particles, lipid-polymernanoparticles, spheroidal nanoparticles, cuboidal nanoparticles,pyramidal nanoparticles, oblong nanoparticles, cylindricalnanoparticles, or toroidal nanoparticles.
 49. The dosage form of claim48, wherein the first synthetic nanocarriers comprise one or morepolymers. 50-54. (canceled)
 55. A method comprising: administering thedosage form of claim 1 to a subject.
 56. The method of claim 55, whereinthe subject has or is at risk of having an infection or infectiousdisease.
 57. The method of claim 55, wherein the subject has or is atrisk of having cancer. 58-64. (canceled)